Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event.
(25/12477)
Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells. (+info)
Regional electrophysiological effects of hypokalaemia, hypomagnesaemia and hyponatraemia in isolated rabbit hearts in normal and ischaemic conditions.
(26/12477)
OBJECTIVE: The aims of this study were to establish an isolated working heart model for electrophysiological recordings from the epicardium and endocardium and to examine regional effects of changes in ion concentrations in normal and ischaemic conditions. METHODS: Monophasic action potential duration (MAPD90), effective refractory period (ERP) and conduction delay were measured simultaneously in the epicardium and endocardium of rabbit hearts paced at 3.3 Hz, subjected to 30 min of regional ischaemia and 15 min of reperfusion. The hearts were exposed before and throughout ischaemia and reperfusion to hypokalaemia (K+ = 2 mM), hypomagnesaemia (Mg2+ = 0.5 mM) or hyponatraemia (Na+ = 110 mM). RESULTS: In the control hearts, no regional electrophysiological differences were seen before ischaemia, but ischaemia-induced MAPD90 shortening and postrepolarisation refractoriness were greater in the epicardium than in the endocardium and conduction delay increased only in the epicardium. Hypokalaemia shortened ERP in the epicardium (but not endocardium) and increased conduction delay in all areas before ischaemia, but it had no effects during ischaemia. During reperfusion hypokalaemia increased the incidence of recurrent tachyarrhythmias. Hypomagnesaemia had no effect before ischaemia, increased epicardial (but not endocardial) MAPD90 shortening during ischaemia, although it had no pro-arrhythmic action. Hyponatraemia increased conduction delay in all areas before ischaemia and produced asystole or severe bradycardia in all hearts. During ischaemia, hyponatraemia decreased ERP shortening and inducibility of arrhythmias in the epicardium (but not endocardium). CONCLUSIONS: We conclude that the more pronounced effect of ischaemia upon the epicardium than the endocardium can be explained by the contact of the endocardium with intracavitary perfusate. We also conclude that changes in ion concentrations may have differential regional electrical effects in normal or ischaemic conditions. (+info)
Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding.
(27/12477)
Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding. (+info)
Dietary magnesium, not calcium, regulates renal thiazide receptor.
(28/12477)
This study reports for the first time a relationship between dietary Mg and the renal thiazide-sensitive Na-Cl cotransporter (TZR, measured by saturation binding with 3H-metolazone). Ion-selective electrodes measured plasma ionized magnesium (PMg++), calcium (PCa++), and potassium (PK+). Restricting dietary Mg for 1 wk decreased PMg++ 18%, TZR 25%, and renal excretion of magnesium (UMg) and calcium (UCa) more than 50% without changing PCa++, PK+, or plasma aldosterone. A low Mg diet for 1 d significantly decreased PMg++, TZR, UMg and UCa. Return of dietary Mg after 5 d of Mg restriction restored PMg++ and TZR toward normal. In the control, Mg-deficient, and Mg-repleting animals, TZR correlated with PMg++ (r = 0.86) and with UMg (r = 0.87) but not UCa (r = 0.09). Increasing oral intake of Mg for 1 wk increased PMg++ 14%, TZR 32%, UMg 74%, and UCa more than fourfold without changing PCa++ or PK+. In contrast, increasing dietary Ca content from 0.02% to 1.91% did not change TZR, but increased UCa fivefold without changing PCa++. Hormonal mediators (if any) involved in the relationship between dietary Mg and TZR remain to be elucidated, as does the relationship between TZR and tubular reabsorption of Mg. (+info)
Intravenous magnesium reduces infarct size after ischemia/reperfusion injury combined with a thrombogenic lesion in the left anterior descending artery.
(29/12477)
Experimental studies have demonstrated that intravenous magnesium (Mg) can protect the ischemic myocardium and has an antithrombotic effect. In patients with myocardial infarction, the reperfusion injury is complicated by the presence of a thrombogenic area in the affected coronary artery that may cause repetitive thrombus formation and embolization. We investigated the effect of Mg on infarct size in a randomized study in pigs. Myocardial infarction was induced by a 50-minute mechanical occlusion of the left anterior descending artery combined with an arterial injury, which stimulated a dynamic thrombus formation with emboli shedding on reperfusion. Magnesium sulfate (6 mmol/20 min plus 3 mmol/h) or saline was started at 30 minutes after coronary occlusion. Real-time ventricular pressure-volume loops were generated from the left ventricle by using a microtip pressure manometer and a conductance catheter. Platelet accumulation in the myocardium was evaluated by using 111In-labeled platelets. After 4 hours of reperfusion, the infarct size/area at risk ratio in the placebo group was 46+/-0.06% (n=8) compared with 22+/-0.07% (n=6) in the Mg-treated animals (P=0. 03). Ejection fraction decreased significantly in the control group but not in the Mg-treated animals (P=0.03). Platelet accumulation in the myocardium did not change significantly between the Mg- and placebo-treated animals (placebo group, 191+/-19%; Mg group, 177+/-29%; NS). The present study demonstrates that intravenous Mg infusion is able to reduce infarct size by >50% and preserve the ejection fraction in this model where ischemia/reperfusion injury was evaluated in the presence of a thrombogenic area in the nutrient artery. (+info)
hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA.
(30/12477)
Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches. (+info)
Women's dietary intakes in the context of household food insecurity.
(31/12477)
A study of food insecurity and nutritional adequacy was conducted with a sample of 153 women in families receiving emergency food assistance in Toronto, Canada. Contemporaneous data on dietary intake and household food security over the past 30 d were available for 145 of the women. Analyses of these data revealed that women who reported hunger in their households during the past 30 d also reported systematically lower intakes of energy and a number of nutrients. The effect of household-level hunger on intake persisted even when other economic, socio-cultural, and behavioral influences on reported dietary intake were considered. Estimated prevalences of inadequacy in excess of 15% were noted for Vitamin A, folate, iron, and magnesium in this sample, suggesting that the low levels of intake associated with severe household food insecurity are in a range that could put women at risk of nutrient deficiencies. (+info)
ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits.
(32/12477)
Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I-D-ATP complex. The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I-D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803. We demonstrate the purification of an I-D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5'-[gamma-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-imido]triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI-ChlD complex. A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its own, whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed. (+info)