Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis.

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria.

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD(133) and DQLD(149), cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.

Why use Finapres or Portapres rather than intra-arterial or intermittent non-invasive techniques of blood pressure measurement?

In the clinic, blood pressure is measured almost exclusively using non-invasive intermittent techniques, of which the auscultatory (Riva-Rocci/Korotkoff, RRK) and the computerized oscillometric method are most often used. However, both methods only provide a momentary value. In addition, the accuracy is hampered by phenomena such as cuff response and white coat hypertension, thus providing artefactually increased values. The vascular unloading technique of Penáz together with the Physiocal criteria of Wesseling provide reliable, non-invasive and continuous estimates of blood pressure. This technique is thus an alternative to the invasive intra-arterial measurements in many cases, without the risks and ethical questions inherent to invasive measurements. Since the pressure waveform is available continuously, computations such as pulse contour and Modelflow cardiac output, spectral analysis and baroreflex sensitivity provide further information on the dynamics of the cardiovascular system on a beat-to-beat basis, similar to intra-arterial measurements.

Safety of dobutamine-atropine stress echocardiography in patients with suspected or proven coronary artery disease.

The purpose of this study was to establish the safety of high-dose dobutamine-atropine stress echocardiography in patients with suspected or proven coronary artery disease. Six hundred fifty consecutive examinations were completed. Mean age of patients was 61 years; 300 had a previous myocardial infarction. Heart rate increased from 73 to 129 beats/min during stress testing, blood pressure did not change significantly (from 140/81 to 150/80 mm Hg). Atropine was added to dobutamine in 239 patients when no ischemia was induced with dobutamine alone and the peak heart rate was < 85% of the theoretical maximal heart rate. Atropine was more frequently administered to patients taking beta blockers (77 vs 27%, p < 0.001). New wall motion abnormalities developed in 243 patients (37%). Significant or symptomatic cardiac tachyarrhythmias, or both, developed during 24 examinations: 1 patient developed ventricular fibrillation, 3 patients developed sustained ventricular tachycardia, 12 patients experienced nonsustained ventricular tachycardia (< 10 beats) and 8 patients had paroxysmal atrial fibrillation. Cardiac arrhythmias were more frequent in patients with a history of ventricular arrhythmias (ventricular tachycardia and fibrillation) (odds ratio 9.9, 2.0 to 45) or left ventricular dysfunction at rest (wall motion score > 1.12) (odds ratio 2.9, 1.1-7.6), but not associated with atropine addition. No death or myocardial infarction occurred. The full dose was not given to 13 patients despite absence of signs or markers of ischemia for limiting side effect, yielding an overall feasibility of the stress test of 98%.(ABSTRACT TRUNCATED AT 250 WORDS)

Ablation of ventricular tachycardia of right ventricular origin with low energy shocks.

Three shocks of 2 joules (2,000 volts within 0.3 msec) were given to a patient with ventricular tachycardia of right ventricular origin at the site of the earliest activation. A standard 6 Fr USCI mapping catheter was used. After the first shock the cycle length prolonged from 385 to 531 msec. After the second shock, the tachycardia was no longer inducible and it remained so after 1 week. No recurrences were seen during a follow-up period of 8 months. This is the first report demonstrating efficacy of modified low energy shocks for ventricular tachycardia.

[A study of the causes of staghorn calculi]. / La recherche des causes du calcul coralliforme.

A thorough metabolic evaluation of all staghorn stone patients seems justified, considering the results obtained by the study of 27 such cases. Pak's ambulatory screening test, slightly modified, was used. This allowed the finding of a hypercalciuria in more than 50% of the cases, a hyperuricosuria in 63% of the cases and a hyperoxaluria in one case out of five. A metabolic anomaly was not detected in two patients. Although urinary tract infection, present in 75% of the cases is essential to the genesis of a staghorn stone, the question raises whether metabolic anomalies are not the primary cause of the stone formation.

Captopril: a protective agent in renal warm ischemia in rats.

Captopril, an angiotensin-converting enzyme inhibitor, was used as a protective agent before and after 90 min of renal warm ischemia in rats. At a dose of 1 mg/kg, it was able to lower the postoperative serum creatinine values significantly (from 6.3 to 2.5 mg% on the 2nd postoperative day) in unilaterally nephrectomized animals. Increasing the dose to 4 and 8 mg/kg or combining captopril with hypertonic mannitol (10 ml/kg of a 15% aqueous solution) did not improve these figures significantly. The same magnitude of protection was obtained when captopril was given either before or just after the ischemia. In terms of mortality the differences among unprotected and protected groups were very important: only 6 of 24 unprotected animals survived the 5th postoperative day while 42 of 44 protected animals survived.