Plasma membrane K ATP channels are highly sensitive to the family of drugs known as K ؉ channel openers, raising the question whether mitochondrial K ATP channels are similarly sensitive to these agents. We addressed this question by measuring K ؉ flux in intact rat liver mitochondria and in liposomes containing K ATP channels purified from rat liver and beef heart mitochondria. K ؉ channel openers completely reversed ATP inhibition of K ؉ flux in both systems. In liposomes, ATP-inhibited K ؉ flux was restored by diazoxide (K1 ⁄2 ‫؍‬ 0.4 M), cromakalim (K1 ⁄2 ‫؍‬ 1 M), and two developmental cromakalim analogues, EMD60480 and EMD57970 (K1 ⁄2 ‫؍‬ 6 nM). Similar K1 ⁄2 values were observed in intact mitochondria. These potencies are well within the range observed with plasma membrane K ATP channels. We also compared the potencies of these K ؉ channel openers on the plasma membrane K ATP channel purified from beef heart myocytes. The K ATP channel from cardiac mitochondria is 2000-fold more sensitive to diazoxide than the channel from cardiac sarcolemma, indicating that two distinct receptor subtypes coexist within the myocyte. We suggest that the mitochondrial K ATP channel is an important intracellular receptor that should be taken into account in considering the pharmacology of K ؉ channel openers.K ϩ channel openers (KCOs) 1 activate ATP-inhibited K ATP channels. As described in several excellent reviews (1-3), members of this drug family exhibit a rich and clinically important pharmacology. Thus, cell membrane K ATP channels (cellK ATP ) in different tissues are considered to mediate the hypotensive and diabetogenic effects of diazoxide (4) and the cardioprotective effects of cromakalim and its derivatives (5). It is important to determine whether these drugs also act on mitochondrial K ATP channels (mitoK ATP ) in their therapeutic range.In the first reports of KCO actions in mitochondria, Belyaeva et al. (6) and Szewczyk et al. (7) observed stimulation of K ϩ uptake by KCOs in respiring mitochondria. RP66471 was the most potent KCO studied (K1 ⁄2 ϭ 50 M), whereas P1060 and diazoxide were only weakly active at 700 M. Because these concentrations are much higher than K1 ⁄2 values observed with cellK ATP (1), these results appear to imply that mitochondrial actions of KCOs are not pharmacologically important.We now report that diazoxide, cromakalim, and two experimental benzopyran derivatives are very potent activators of K ϩ flux through ATP-inhibited mitoK ATP , with K1 ⁄2 values similar to those observed with cellK ATP . KCO activation of K ϩ flux was observed in both intact mitochondria and proteoliposomes containing reconstituted mitoK ATP . No effect was observed on uninhibited K ϩ flux, which likely explains the low potencies observed by previous workers (6, 7) in assays that did not include Mg 2ϩ and ATP. We also found that mitoK ATP and cellK ATP from beef heart differed strongly in their sensitivity to diazoxide, indicating distinct receptor subtypes among K ATP channels from the same cell. Our result...

Polymeric nanoparticles with glucose-responsiveness under physiological conditions are of great interests in developing drug delivery system for the treatment of diabetes. Herein, glucose-responsive complex micelles were prepared by self-assembly of a phenylboronic acid-contained block copolymer PEG-b-P(AA-co-APBA) and a glycopolymer P(AA-co-AGA) based on the covalent complexation between phenylboronic acid and glycosyl. The formation of the complex micelles with a P(AA-co-APBA)/P(AA-co-AGA) core and a PEG shell was confirmed by HNMR analysis. The glucose-responsiveness of the complex micelles was investigated by monitoring the light scattering intensity and the fluorescence (ARS) of the micelle solutions. The complex micelles displayed an enhanced glucose-responsiveness compared to the simple PEG-b-P(AA-co-APBA) micelles and the sensitivity of the complex micelles to glucose increased with the decrease of the amount of P(AA-co-AGA) in the compositions. The cytotoxicity of the polymers and the complex micelles was also evaluated by MTT assay. This kind of complex micelles may be an excellent candidate for insulin delivery and may find application in the treatment of diabetes.

Phenylboronic acid (PBA)-based polymers have attracted much attention because of their potential applications in glucose-responsive insulin delivery for the treatment of diabetes. Herein, we prepared a kind of glucose-responsive complex micelles by the complexation between a phenylboronic acidcontaining block copolymer PEG-b-P(Asp-co-AspPBA) and a glycopolymer P(Asp-co-AGA). The complex micelles combined a variety of advantages such as stability against aggregation due to the PEG shell, fast response to glucose at neutral pH due to the complex core composed of two hydrophilic polymers, and more importantly better glucose sensitivity ascribed to the decreased apparent pK a of the PBA/AGA complex. Moreover, glucose-triggered on-off release of insulin was obtained under physiological pH 7.4 with 2 g L À1 glucose (hyperglycemia), which provided us with an effective strategy for self-regulated insulin delivery in response to physiological glucose level. The enhanced biocompatibility of this complex micelles system was confirmed by MTT assay. This type of complex micelles may be a promising candidate for in vivo insulin delivery.

The bovine genetic resources in China are diverse, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Chinese cattle, we analysed the whole genomes of 46 cattle from six phenotypically and geographically representative Chinese cattle breeds, together with 18 Red Angus cattle (RAN) genomes, 11 Japanese black cattle (JBC) genomes and taurine and indicine genomes available from previous studies. Our results showed that Chinese cattle originated from hybridization between Bos taurus and Bos indicus. Moreover, we found that the level of genetic variation in Chinese cattle depends upon the degree of indicine content. We also discovered many potential selective sweep regions associated with domestication related to breed-specific characteristics, with selective sweep regions including genes associated with coat colour (ERCC2, MC1R, ZBTB17 and MAP2K1), dairy traits (NCAPG, MAPK7, FST, ITFG1, SETMAR, PAG1, CSN3 and RPL37A), and meat production/quality traits (such as BBS2, R3HDM1, IGFBP2, IGFBP5, MYH9, MYH4 and MC5R). These findings substantially expand the catalogue of genetic variants in cattle and reveal new insights into the evolutionary history and domestication traits of Chinese cattle.

The mitochondrial K ATP channel (mitoK ATP ) is highly sensitive to ATP, which inhibits K ؉ flux with K1 ⁄2 values of 20 -40 M. This raises the question, how can mitoK ATP be opened in the presence of physiological concentrations of ATP? We measured K ؉ flux in liposomes reconstituted with purified mitoK ATP and found that guanine nucleotides are potent activators of this channel. ATPinhibited K ؉ flux was completely reactivated by both GTP (K1 ⁄2 ‫؍‬ 7 M) and GDP (K1 ⁄2 ‫؍‬ 140 M). These ligands had no effect in the absence of ATP. (1) reported electrophysiological evidence from patch clamp studies of fused mitoplasts, and we described reconstitution of a highly purified mitoK ATP (2, 3). In our protocols, which have the advantage of being free of the complexities of intact mitochondria, K ϩ flux is measured using steady-state spectroscopy of the fluorescent probe PBFI. These measurements have permitted initial characterization of the kinetics and regulation of mitoK ATP (4). The K m for K ϩ is 32 mM, and the channel is highly selective for K ϩ (Na ϩ and TEA ϩ are neither transported nor do they affect K ϩ flux through mitoK ATP ). MitoK ATP is inhibited with high affinity by ATP and ADP, and this inhibition exhibits an absolute requirement for divalent cations. We have recently shown that ATP inhibition of K ϩ flux through mitoK ATP is reversed by submicromolar levels of K ϩ channel openers (5). We also demonstrated mitoK ATP activity in respiring rat liver mitochondria (6). The confounding and unavoidable coexistence of K ϩ diffusion (leak) was controlled by comparing K ϩ flux to TEA ϩ flux, for which there are no endogenous pathways other than diffusive leak. We made the simple demonstration that ATP inhibited K ϩ uptake to rates similar to those of TEA ϩ uptake and had no effect on TEA ϩ uptake itself. These studies left us with a conundrum: given the high affinity for ATP, how can mitoK ATP ever be opened under normal physiological conditions? We hypothesized (4) that endogenous activators of mitoK ATP must exist to overcome the high affinity for ATP, and we now present support for this hypothesis. K ϩ flux through the MgATP-inhibited channel is restored to full activity by GTP and GDP, neither of which has any effect in the absence of MgATP. GTP and GDP are competitive with ATP, and their reversal of ATP inhibition exhibits hyperlinear kinetics consistent with two guanine nucleotide binding sites. We also report that palmitoyl-CoA and oleoylCoA inhibit mitoK ATP with high potency, and this inhibition is also reversed by GTP and by the potassium channel openers, cromakalim and diazoxide. Inhibition by long-chain acyl-CoA esters, like inhibition by ATP, exhibits an absolute requirement for Mg 2ϩ ions and is immediately reversed upon chelation of Mg 2ϩ . From these findings, we infer that GTP and longchain acyl-CoA esters may be the physiological regulators of mitoK ATP and that this channel may play a role in vivo in regulating fatty acid oxidation. EXPERIMENTAL PROCEDURES Extraction, Purification, and Reco...

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e VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4B (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorageindependent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronichypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer.