Is reduced SERCA2a expression detrimental or beneficial to postischemic cardiac function and injury? Evidence from heterozygous SERCA2a knockout mice

Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.     

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.     

Structural analysis of 2D crystals of gastric H+,K+-ATPase in different states of the transport cycle

The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1PADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E1 to the E1P x ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E1 and E1P x ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E1 and E1P x ADP states present in the 2D crystals,showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase.     

Junction-forming aquaporins

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.     

Association of a polymorphism of ABCB1 with obesity in Japanese individuals

The aim of the present study was to identify gene polymorphisms that confer susceptibility to obesity. A total of 5448 unrelated Japanese individuals from two independent populations were examined: subject panel A comprised 4252 individuals who visited participating hospitals; subject panel B comprised 1196 community-dwelling elderly individuals. The genotypes for 95 polymorphisms of 67 candidate genes were determined. The χ² test revealed that six polymorphisms were related (p < 0.05) to the prevalence of obesity in subject panel A; after application of Bonferroni's correction, however, only the 2677G → A/T polymorphism (rs2032582) of the ATP-binding cassette, subfamily B, member 1 gene (ABCB1) was significantly associated (p = 0.0003) with obesity. Subsequent multivariable logistic regression analysis also revealed that the 2677G → A/T polymorphism of ABCB1 was significantly associated with obesity. For validation of this association, the 2677G → A/T polymorphism of ABCB1 was examined in subject panel B and again found to be significantly associated with obesity. Body mass index was significantly (p = 0.01) greater for individuals with the variant T allele of this polymorphism than for those with the GG genotype in the combined subject panels A and B. Our results suggest that the ABCB1 genotype may prove informative for assessment of genetic risk for obesity in Japanese individuals.     

Cycloprodigiosin hydrochloride activates the Ras-PI3K-Akt pathway and suppresses protein synthesis inhibition-induced apoptosis in PC12 cells

Cycloprodigiosin hydrochloride (cPrG-HCl), a member of the prodigiosin family of compounds, has been reported to act as an H(+)/Cl(-) symporter. This compound induces apoptosis in several cancer cells and acts as an antitumor drug in animal models. In this study, we found a novel function of cPrG-HCl; to suppress cell death in PC12 cells, which is caused by protein synthesis inhibitors cycloheximide and actinomycin D. cPrG-HCl activated Akt and suppressed apoptosis, and this was accompanied by inhibition of caspase-3 activity and DNA fragmentation independently of its H(+)/Cl(-) symporter activity. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and dominant-negative Ras attenuated the anti-apoptotic activity of cPrG-HCl, which indicates that cPrG-HCl activated the Ras-PI3K-Akt pathway suppressing apoptosis. On the other hand, serum-deprivation-induced apoptosis was not suppressed by cPrG-HCl.     

Generation of human monoclonal antibodies against <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> by applying the phage display method to human peripheral blood mononuclear cells immunized in vitro

Propionibacterium acnes is a gram-positive, non-spore-forming, rod-shaped bacterium that is often detected in normal human skin flora. P. acnes has been associated with many diseases. In this study, we attempted to generate anti-P. acnes human monoclonal antibodies. A phage antibody library was first generated from human peripheral blood mononuclear cells immunized in vitro with P. acnes using the phage display method, and P. acnes-specific phage antibodies were obtained using solid phase panning. Antigen-specific variable region genes were then amplified and recombined into vectors expressing human IgG antibodies. The results indicated that the recombinant human IgG antibodies exhibited P. acnes-specific binding. This study demonstrates that the combined use of an in vitro immunization protocol and the phage display method enables the generation of human monoclonal antibodies against pathogenic bacteria and toxic antigens.