Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

(Ni-Sn-M_xO_y)/Pb改性复合电极电解合成L-半胱氨酸的反应

在（Ｎｉ－Ｓｎ）／Ｐｂ合金修饰电极中添加某些金属氧化物进行改性，用于电解合成Ｌ－半胱氨酸反应。结果证明，添加ＷＯ３后电极反应活性大大加强，反应同期转化率有较大提高，选择性也有所提高，添加稀土金属氧化物能有效降低反应阴极过电位，使反应选择性有所提高，但是电极稳定性还有待改善。     

Dietary fat influences electric membrane properties of neurons in cell culture

1. SJL/J mice were maintained on semipurified diets which differed in the ratio of polyunsaturated/saturated fatty acid content (P/S). Exposure was from conception and was maintained for periods ranging from 6 to 34 weeks. 2. Neural cell cultures were prepared from dorsal root ganglia (DRG). After 6 and 20 days of culture, neuronal electric membrane properties were determined quantitatively by intracellular recording. 3. A number of significant differences were observed for the two dietary conditions. DRG from mice on the low-P/s diet had an increase in the rate of fall of both phases of repolarization which, in conjunction with the reduced action potential overshoot, led to a reduced action potential duration. This shift to shorter-duration action potentials was accompanied by a shift to more monophasic falling phases. The low-P/S neurons also exhibited a decreased afterhyperpolarization, decreased specific membrane resistance, and decreased membrane electrical time constant compared to high-P/S neurons. 4. It was concluded that the P/S ratio in the diet can have a significant effect on the electric properties of neurons. The high-P/S neurons tended to have action potentials with biphasic repolarizations and longer durations. In contrast, the low-P/S neurons tended to have action potentials with monophasic repolarizations and shorter durations. Moreover, the known ionic dependence of these two types of action potentials suggested that the low-P/S diet resulted in action potentials with a more exclusive Na dependence, while the high-P/S diet resulted in action potentials with both Na and Ca dependence.     

Determination of membrane potential with lipophilic cations: correction of probe binding

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–4) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, C_i^app is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.     

Electrolysis-mediated irreversible inactivation of lipoxygenase directed toward electroaffinity labelling

Irreversible inhibition of soybean lipoxygenase-1 (SL-1) was accomplished via a controlled potential oxidative electrolysis of 1,5-dihydroxynaphthalene (1,5-DHN) at +0.8 V vs SCE. The inactivation of SL-1 with this known inhibitor was greatly enhanced under these electrolytic conditions to which the enzyme itself was stable. Electrolyses were run at 0 degree C in a 0.05 M phosphate buffer, pH 7.0, using graphite cloth electrodes. The rate of inactivation was observed to be limited by and dependent on the anodic oxidation of 1,5-DHN. The non-oxidizable (at this potential) inhibitor indomethacin was shown to protect the enzyme from irreversible inactivation, however, an external nucleophile (2-mercaptoethanol) had little effect. These initial studies support the capability of such electrochemical methods for the site-specific covalent modification (affinity labelling) of lipoxygenase, and perhaps other enzymes.     

A practical approach for quantitating specific mRNAs by solution hybridization

The preparation and use of a specific cDNA probe for quantitating mRNA by solution hybridization is described. Cloned DNA sequences are nick translated, denatured, hybridized to single-stranded M13 clones containing message strand (mDNA) sequences, and separated chromatographically on Bio-Gel A50 under first native and then denaturing conditions to yield a single-stranded cDNA probe. The details of a solution hybridization assay in which the single-stranded cDNA is used to quantitate mRNA in total nucleic acid samples are described. As little as 0.5 pg of mRNA can easily be detected within a day of sample isolation. Thus, the assay is both rapid and sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. It is ideally suited to situations when accurate quantitation of multiple samples is anticipated.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

细胞色素c在氨基酸及二肽修饰金电极上的电化学反应

系统地研究了细胞色素ｃ在多种氨基酸和多肽修饰电极上的电化学反应。并对影响加速细胞色素ｃ电化学反应的因素进行了讨论。