Synthesis, magnetic properties and MCD spectra of a four coordinate copper(II) complex with two chelated phenolate-substituted imino nitroxides

A reaction of Cu(II) nitrate and 4,4,5,5-tetramethyl-2-(2-hydroxophenyl)imidazolin-1-oxyl (IM2PhOH) with potassium methoxide in methanol gave a homoleptic bis(imino nitroxide) complex of [Cu(IM2PhO)₂] (1). The single-crystal X-ray analysis of 1 showed that the imino nitroxide anion, IM2PhO⁻, chelated to a Cu^II ion via an imino-N and a phenoxide-O atoms to form a six-membered chelate ring. The coordination geometry around the Cu(II) ion was a distorted square-planar polygon; the dihedral angle between the two coordination planes, each of which was defined by Cu and two ligating atoms of IM2PhO⁻, was 40.81°. The temperature dependences of the magnetic susceptibility and the EPR spectra of 1 indicate that the magnetic interaction between Cu(II) and the imino nitroxide is ferromagnetic, while there is a moderate antiferromagnetic interaction between two coordinated imino nitroxides. A balance between these opposite interactions attains the lowest molecular doublet spin-state in 1. The variable temperature magnetic circular dichroism (MCD) spectrum of the complex 1 also showed two negative components with a large C term, which may be due to the charge-transfer (CT) transition originated from the d orbital to the SOMO π^* orbital in the spin-coupled IM2PhO⁻ radicals; resulting in the largely split doublet excited states with the spin singlet and triplet d⁸ configurations.     

Simultaneous determination of three local anesthetic drugs from the pipecoloxylidide group in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C(18) reversed-phase column (150 x 4.6 mm I.D.) filled with 5-microm particles and attached to a UV detector. The mobile phase was composed of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88-91% for mepivacaine, 87-89% for ropivacaine and 88-91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.     

Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.     

A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A-H2B, we identified protein phosphatase (PP) 2C gamma subtype (PP2Cgamma/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A-H2B. The disruption of PP2Cgamma in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cgamma-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.     

Nardilysin enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of tumor necrosis factor-alpha-converting enzyme

Like other members of the epidermal growth factor family, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a transmembrane protein that can be shed enzymatically to release a soluble growth factor. Ectodomain shedding is essential to the biological functions of HB-EGF and is strictly regulated. However, the mechanism that induces the shedding remains unclear. We have recently identified nardilysin (N-arginine dibasic convertase (NRDc)), a metalloendopeptidase of the M16 family, as a protein that specifically binds HB-EGF (Nishi, E., Prat, A., Hospital, V., Elenius, K., and Klagsbrun, M. (2001) EMBO J. 20, 3342-3350). Here, we show that NRDc enhances ectodomain shedding of HB-EGF. When expressed in cells, NRDc enhanced the shedding in cooperation with tumor necrosis factor-alpha-converting enzyme (TACE; ADAM17). NRDc formed a complex with TACE, a process promoted by phorbol esters, general activators of ectodomain shedding. NRDc enhanced TACE-induced HB-EGF cleavage in a peptide cleavage assay, indicating that the interaction with NRDc potentiates the catalytic activity of TACE. The metalloendopeptidase activity of NRDc was not required for the enhancement of HB-EGF shedding. Notably, a reduction in the expression of NRDc caused by RNA interference was accompanied by a decrease in ectodomain shedding of HB-EGF. These results indicate the essential role of NRDc in HB-EGF ectodomain shedding and reveal how the shedding is regulated by the modulation of sheddase activity.     

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.     

The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model

Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway.     

Exogenous and endogenous ghrelin counteracts GLP-1 action to stimulate cAMP signaling and insulin secretion in islet β-cells

We studied interactive effects of insulinotropic GLP-1 and insulinostatic ghrelin on rat pancreatic islets. GLP-1 potentiated glucose-induced insulin release and cAMP production in isolated islets and [Ca(2+)](i) increases in single β-cells, and these potentiations were attenuated by ghrelin. Ghrelin suppressed [Ca(2+)](i) responses to an adenylate cyclase activator forskolin. Moreover, GLP-1-induced insulin release and cAMP production were markedly enhanced by [D-lys(3)]-GHRP-6, a ghrelin receptor antagonist, in isolated islets. These results indicate that both exogenous and endogenous islet-derived ghrelin counteracts glucose-dependent GLP-1 action to increase cAMP production, [Ca(2+)](i) and insulin release in islet β-cells, positioning ghrelin as a modulator of insulinotropic GLP-1.