Phosphorus magnetic resonance spectroscopy: its utility in examining the membrane hypothesis of schizophrenia

A novel approach to understanding the pathophysiology of schizophrenia has been the investigation of membrane composition and functional perturbations, referred to as the "Membrane Hypothesis of Schizophrenia." The evidence in support of this hypothesis has been accumulating in findings in patients with schizophrenia of reductions in phospholipids and essential fatty acids various peripheral tissues. Postmortem studies indicate similar reductions in essential fatty acids in the brain. However, the use of magnetic resonance spectroscopy (MRS) has provided an opportunity to examine aspects of membrane biochemistry in vivo in the living brain. MRS is a powerful, albeit complex, noninvasive quantitative imaging tool that offers several advantages over other methods of in vivo biochemical investigations. It has been used extensively in investigating brain biochemistry in schizophrenia. Phosphorus MRS (31P MRS) can provide important information about neuronal membranes, such as levels of phosphomonoesters that reflect the building blocks of neuronal membranes and phosphodiesters that reflect breakdown products. 31P MRS can also provide information about bioenergetics. Studies in patients with chronic schizophrenia as well as at first episode prior to treatment show a variety of alterations in neuronal membrane biochemistry, supportive of the membrane hypothesis of schizophrenia. Below, we will briefly review the principles underlying 31P MRS and findings to date. Magnetic resonance spectroscopy (MRS) is a powerful, albeit complex, imaging tool that permits investigation of brain biochemistry in vivo. It utilizes the magnetic resonance imaging hardware. It offers several advantages over other methods of in vivo biochemical investigations. MRS is noninvasive, there is no radiation exposure, does not require the use of tracer ligands or contrast media. Because of it is relatively benign, repeated measures are possible. It has been used extensively in investigating brain biochemistry in schizophrenia.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.     

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.     

颅内静脉系统血栓形成6 例临床分析

目的:探讨颅内静脉系统血栓形成(CVT)的临床表现、影像学特征以及治疗方法对临床诊断的意义。方法:回顾性分析首都医科大学宣武医院收治的6例CVT患者的临床表现及影像学特征与治疗方法。结果:6例临床表现无特殊,4例经MRI+MRV确诊,3例DSA检查确诊,1例介入治疗,4例抗凝治疗,2例保守,无死亡患者。结论:静脉系统血栓形成临床症状缺乏特异性,临床遇到急性起病的头痛、呕吐,伴或不伴有局灶性神经功能缺损或癫痫发作、意识障碍的青中年人,应高度怀疑CVT。早期应用抗凝、溶栓等治疗方法,对改善预后具有较高的临床应用价值。     

Impact of exogenous lysolipids on sensitive and multidrug resistant K562 cells: 1H NMR studies

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.