Iron overload adversely affects outcome of allogeneic hematopoietic cell transplantation.

Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT) for hematologic disorders. Serum ferritin, a marker of tissue iron overload, was measured immediately before transplant in adult patients undergoing myeloablative HCT from matched sibling or unrelated donors. The effect of elevated pretransplant ferritin (defined as ferritin >or=1000 ng/ml) on day 100 mortality, overall survival, acute GVHD and infectious complications was assessed. Data on 190 patients were analyzed. In univariate analysis, the high-ferritin group had increased day 100 mortality (20 vs 9%, P=0.038), decreased overall survival (log-rank test: P-value=0.004), increased acute GVHD/death (63 vs 43%, P=0.009) and increased incidence of blood stream infections (BSIs)/death (60 vs 44%, P=0.042). In a multivariate analysis, high ferritin was associated with increased risk of death (Cox model: hazard ratio=2.28, P=0.004), increased day 100 mortality (generalized linear model (GLM) odds ratio=3.82, P=0.013), increased incidence of acute GVHD/death (GLM odds ratio=3.11, P=0.001) and increased risk of BSI/death (GLM odds ratio=1.99, P=0.032). The results remained similar when serum ferritin was considered a continuous variable. Elevated serum ferritin adversely impacts on overall survival and increases the likelihood of acute GVHD and BSI after allogeneic HCT.

Mechanism for nucleotide exchange in monomeric actin.

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Divalent cation binding to the high- and low-affinity sites on G-actin.

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.