Effect of dimethyl adipimidate on K+ transport and shape change in red blood cells from sickle cell patients.
(1/18)
Dimethyl adipimidate (DMA) reduces K+ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (Psickle), the Ca2+-activated K+ channel (or Gardos channel) and the K+-CI- cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca2+ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of Psickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K+ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease. (+info)
A genomic approach to the identification and characterization of HOXA13 functional binding elements.
(2/18)
HOX proteins are important transcriptional regulators in mammalian embryonic development and are dysregulated in human cancers. However, there are few known direct HOX target genes and their mechanisms of regulation are incompletely understood. To isolate and characterize gene segments through which HOX proteins regulate transcription we used cesium chloride centrifugation-based chromatin purification and immunoprecipitation (ChIP). From NIH 3T3-derived HOXA13-FLAG expressing cells, 33% of randomly selected, ChIP clones were reproducibly enriched. Hox-enriched fragments (HEFs) were more AT-rich compared with cloned fragments that failed reproducible ChIP. All HEFs augmented transcription of a heterologous promoter upon coexpression with HOXA13. One HEF was from intron 2 of Enpp2, a gene highly upregulated in these cells and has been implicated in cell motility. Using Enpp2 as a candidate direct target, we identified three additional HEFs upstream of the transcription start site. HOXA13 upregulated transcription from an Enpp2 promoter construct containing these sites, and each site was necessary for full HOXA13-induced expression. Lastly, given that HOX proteins have been demonstrated to interact with histone deacetylases and/or CBP, we explored whether histone acetylation changed at Enpp2 upon HOXA13-induced activation. No change in the general histone acetylation state was observed. Our results support models in which occupation of multiple HOX binding sites is associated with highly activated genes. (+info)
Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.
(3/18)
In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb. (+info)
Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin.
(4/18)
The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method. (+info)
Imidoester inhibition of lymphocyte DNA synthesis.
(5/18)
Imidoesters amidinate free amino groups and produce inter- and intramolecular covalent bonds. To determine whether imidoesters influenced lymphocyte transformation, human peripheral blood or calf lymph node lymphocytes were cultured with dimethyladipimate (DMA), a bifunctional (cross-linking) imidoester, or methyl acetimidate (MAC), a monofunctional (noncross-linking) imidoester. Both DMA and MAC decreased the rate of endogenous DNA synthesis in a dose-dependent fashion. In further work, lymphocytes were treated with Phaseolus vulgaris phytohemagglutinin, concanavalin A, or periodate. DMA (1 mM) decreased DNA synthesis in P. vulgaris phytohemagglutinin-stimulated human cells by 65%, Concanavalin A-stimulated cells by 98.2%, and periodate-stimulated cells by 85%. Similar results were obtained with 1 mM MAC. Inhibition by DMA was slightly greater than was the inhibition by MAC. Decreased DNA synthesis resulted if DMA was added to P. vulgaris phytohemagglutinin-stimulated human lymphocytes at initiation of culture (72%) or after 16 hr (75%); inhibition was less when DMA was added after 24 hr (43%) and was not apparent if added after 48 hr. Therefore, both monofunctional and bifunctional imidoesters inhibit endogenous and stimulated DNA synthesis in human and calf lymphocytes. (+info)
Topographical studies on poliovirus capsid proteins by chemical modification and cross-linking with bifunctional reagents.
(6/18)
Poliovirus capsid proteins comprise 15.1 lysines in VP1, 5.6 lysines in VP2, 11.7 lysines in VP3 and 5.5 lysines in VP4. Treatment with monofunctional reagent N-succinimidyl 2,3-3H-proprionate leads to the modification of 3.4 lysines in VP1, 0.6 lysines in VP2, 2.0 lysines in VP3 and 0.03 lysines in VP4. Chemical modification with the monofunctional reagent N-succinimidyl 3-(4-hydroxy,5-125I-iodophenyl)propionate results in a predominant labelling of VP1 and VP3, whereas VP2 is less accessible and VP4 is not modified. Cross-linking of poliovirus with bifunctional imidoesters, dimethyl suberimidate (DMS, 1.1 nm) and dimethyl adipimidate (DMA, 0.8 nm) leads to a new protein complex of mol. wt. which corresponds to the sum of VP1 and VP3. By cleavage with ammonia and electrophoresis on polyacrylamide gels in SDS, the proteins are identified as VP1 and VP3. This result gives evidence for a direct neighbourhood of VP1 and VP3 in the virus capsid. Treatment of the virus with the mono- and bifunctional reagents has no influence on the stability of the particle. The infectivity is reduced only by the bifunctional reagent. (+info)
Abnormalities in the erythrocyte membrane in acute lymphoid leukaemia.
(7/18)
Erythrocytes from patients suffering from acute lymphoid leukaemia (ALL) show decreased proportions of spectrin tetrameters and altered spatial distribution of band 4.1 and ankyrins. These abnormalities of the cytoskeleton are probably responsible for altered membrane fluidity and transbilayer distribution of phosphatidylethanolamine in ALL. ALL is associated with severe anaemia and usually, but not always, with overproduction of lymphocytes. To our knowledge, this is the first report of abnormalities in the erythrocyte membrane in ALL which may, in part, be responsible for the observed anaemia. (+info)
Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.
(8/18)
Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes. (+info)