The interconversion between monomeric and dimeric bovine heart cytochrome c oxidase

Monomers and dimers of bovine heart cytochrome c oxidase (EC 1.9.3.1.) were separated by gel filtration chromatography on Ultrogel AcA 34 or by sucrose gradient centrifugation. Factors influencing the interconversion of the two aggregation states of this enzyme were analyzed. At very low ionic strength, in the presence of dodecyl maltoside, monomers were the main species. Salts appeared to stabilize the dimeric form, divalent cations being more efficient than monovalent. High enzyme concentrations favoured the formation of dimers, also at low ionic strength. The type of detergent had a strong influence on the monomer-dimer interconversion; in Triton X-100 and dodecyl maltoside (at high ionic strength) cytochrome c oxidase was homogenously dispersed in its dimeric form, while in Tween-80 gel filtration showed only large particles eluting in the void volume. In cholate monomers and aggregates were observed but no dimers. The aggregation state had an influence on the steady state kinetics of the ferrocytochrome c oxidase activity. Monomers showed linear Eadie-Hofstee plots, whilst the dimeric and aggregated enzyme gave nonlinear Eadie-Hofstee plots. Ionic strength, enzyme concentration and type of detergent were affecting the enzyme's kinetics in a way consistent with the molecular form obtained by the gel filtration or sedimentation analysis. The data support a negative cooperative mechanism for the interaction of cytochrome c with the dimeric enzyme, as proposed earlier (K.A. Na?ecz et al., (1983) Biochem. Biophys. Res. Commun., 114, 822-828).     

Transplantation of expanded bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) improves left ventricular function and remodelling after myocardial infarction

Adult bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) exhibit a Sca‐1⁺/Lin^–/CD45^– phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 10⁶) of freshly isolated, non‐expanded VSEL‐SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL‐SCs in the marrow is very low, we examined whether VSEL‐SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n= 11), 1 × 10⁵ enhanced green fluorescent protein (EGFP)‐labelled expanded untreated VSEL‐SCs (group II, n= 7), or 1 × 10⁵ EGFP‐labelled expanded VSEL‐SCs pre‐incubated in a cardiogenic medium (group III, n= 8). At 35 days after myocardial infarction (MI), mice treated with pre‐incubated VSEL‐SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle‐treated controls. In contrast, transplantation of expanded but untreated VSEL‐SCs did not produce appreciable reparative benefits. Scattered EGFP⁺ cells expressing α‐sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)‐1, or von Willebrand factor were present in VSEL‐SC‐treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL‐SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre‐incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL‐SCs for cardiac repair.     

Overexpression of mitogen-activated protein kinase kinase 6 in the heart improves functional recovery from ischemia in vitro and protects against myocardial infarction in vivo

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, alpha B-crystallin (alpha BC), in cultured cardiac myocytes, we examined alpha BC levels in the mouse hearts. The level of alpha BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in alpha BC levels in the mitochondrial fractions of TG mouse hearts but no increase in alpha BC levels in any of the other fractions analyzed. These alterations in alpha BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.     

Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection

Previous studies have indicated that PKC-epsilon is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-epsilon exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-epsilon signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-epsilon signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.     

Graphite as Cointercalation Electrode for Sodium‐Ion Batteries: Electrode Dynamics and the Missing Solid Electrolyte Interphase (SEI)

The intercalation of solvated sodium ions into graphite from ether electrolytes was recently discovered to be a surprisingly reversible process. The mechanisms of this “cointercalation reaction” are poorly understood and commonly accepted design criteria for graphite intercalation electrodes do not seem to apply. The excellent reversibility despite the large volume expansion, the small polarization and the puzzling role of the solid electrolyte interphase (SEI) are particularly striking. Here, in situ electrochemical dilatometry, online electrochemical mass spectrometry (OEMS), a variety of other methods among scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X‐ray diffraction (XRD) as well as theory to advance the understanding of this peculiar electrode reaction are used. The electrode periodically “breathes” by about 70–100% during cycling yet excellent reversibility is maintained. This is because the graphite particles exfoliate to crystalline platelets but do not delaminate. The speed at which the electrode breathes strongly depends on the state of discharge/charge. Below 0.5 V versus Na⁺/Na, the reaction behaves more pseudocapacitive than Faradaic. Despite the large volume changes, OEMS gas analysis shows that electrolyte decomposition is largely restricted to the first cycle only. Combined with TEM analysis and the electrochemical results, this suggests that the reaction is likely the first example of a SEI‐free graphite anode.     

Preparation of monomeric cytochrome C oxidase: its kinetics differ from those of the dimeric enzyme

Bovine cytochrome c oxidase in 0.1% dodecylmaltoside, 50 mM KCl and 10 mM Tris-HCl, pH 7.4 is monodisperse with an apparent Mr 360,000 (dimer) as estimated by filtration on Ultrogel AcA 34. In the absence of added KCl the apparent Mr is 160,000 (monomer). The dimeric enzyme has a high and a low affinity site for cytochrome c; the monomeric, only the high affinity site. The results are consistent with the existence of one active site per monomer, having high affinity for cytochrome c. Since in a dimer the two sites are in close proximity, the binding of the first molecule of cytochrome c to the first site hinders the binding of the second molecule to the second site. The kinetic data fit with a model of homotropic negative cooperativity. The effect of salts on the cytochrome c oxidase kinetics is also present in isolated bovine heart mitochondria.     

MPG静注减轻清醒狗缺血后心肌顿抑

为了解自由基清除剂２巯基丙酰基甘氨酸（ＭＰＧ）能否减轻缺血后心肌顿抑,本文报告了在清醒狗模型中氧自由基清除剂ＭＰＧ对缺血后心肌顿抑的疗效。３９只清醒狗模型阻闭前降支１５ｍｉｎ后再灌注４８ｈ。治疗组（ｎ＝１７）于阻闭前１５ｍｉｎ始静脉给予ＭＰＧ（１００ｍｇ／ｋｇ·ｈ）,共持续６０ｍｉｎ,对照组（ｎ＝２２）给予生理盐水。结果表明,二组缺血区侧支血流、缺血区大小及血液动力学指标无显著差异,而治疗组室壁收缩增厚指数（一种局部心肌功能指标）于再灌注后２、３、４、５、６ｈ明显大于对照组,当侧支血流低于１０％时,改善更明显。指数回归分析结果显示,治疗组侧支血流越低,收缩功能恢复程度越明显。结论,ＭＰＧ可以促进缺血后心肌顿抑的恢复,这种有益的疗效在低侧支血流时更明显。     

Echocardiography-guided percutaneous left ventricular intracavitary injection as a cell delivery approach in infarcted mice

Molecular and Cellular Biochemistry - In the field of cell therapy for heart disease, a new paradigm of repeated dosing of cells has recently emerged. However, the lack of a repeatable cell...