Comprehensive quantification of ceramide species in human stratum corneum

One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatog­raphy-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm × 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.     

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.Photobiologically produced hydrogen gas (H₂) is a clean energy source with the potential to greatly supplement our use of fossil fuels (39). Whereas coal and oil are limited, cyanobacteria and eukaryotic microalgae can use inexhaustible sunlight as the energy source and water as the electron donor to produce H₂ (42). This gas is generated either by hydrogenases (52) or as an inevitable by-product of N₂ fixation by nitrogenases (49). In contrast to the reaction of hydrogenases which is reversible, nitrogenases catalyze the unidirectional production of H₂, although with substantial energy input in the form of ATP (47). Under optimal N₂-fixing conditions: N₂ + 8 e⁻ + 8 H⁺ + 16 ATP → H₂ + 2 NH₃ + 16 (ADP + P_i), whereas, in the absence of N₂ (e.g., under Ar), all electrons are allocated to proton reduction: 2 e⁻ + 2 H⁺ + 4 ATP → H₂ + 4 (ADP + P_i). Thus, one expects to be able to increase the H₂ production activity of nitrogenase by decreasing the electron allocation to N₂ fixation.Nitrogenases are sensitive to inactivation by O₂; however, N₂-fixing cyanobacteria have developed mechanisms to protect these enzymes from photosynthetically generated oxygen (5). Of particular interest, Anabaena (also known as Nostoc) sp. strain PCC 7120 and some other filamentous cyanobacteria respond to combined-nitrogen deprivation by undergoing differentiation in which a subset of cells become heterocysts that provide a microaerobic environment, allowing nitrogenase to function in aerobic culture conditions. The nitrogenase-related (nif) genes are specifically expressed in heterocysts which lack O₂-evolving photosystem II activity and are surrounded by a thick cell envelope composed of glycolipids and polysaccharides that impede the entry of O₂ (56). Vegetative cells perform oxygenic photosynthesis and fix CO₂. Heterocysts obtain carbohydrates from those cells and, in turn, provide them with fixed nitrogen.The molybdenum-containing nitrogenase of Anabaena sp. strain PCC 7120 consists of the Fe protein (encoded by nifH) and the MoFe protein (encoded by nifD and nifK). As in other organisms, the Fe protein is a homodimer containing a single [4Fe-4S] cluster and functions as an ATP-dependent electron donor to the MoFe protein. The latter is an α₂β₂ heterotetramer with each nifD-encoded α subunit coordinating the FeMo cofactor (FeMo-co; MoFe₇S₉X-homocitrate) that binds and reduces substrate, while α plus the nifK-encoded β subunits coordinate the [8Fe-7S] P-cluster (14). Additional nif genes are required for the biosynthesis of the metal clusters and maturation of the enzyme (40). The major nif gene cluster of Anabaena sp. strain PCC 7120 undergoes two rearrangements in the heterocyst to yield nifB-fdxN-nifSUHDK-(1 ORF)-nifENX-(2 ORFs)-nifW-hesAB-fdxH (19).One approach to increase H₂ production by nitrogenase is to enhance the electron flux to proton reduction and away from N₂ reduction. Although replacement of N₂ by Ar is effective for increasing H₂ production, this approach increases the operational cost for large-scale generation of H₂. Mutagenesis offers an alternative mechanism to overcome N₂ competition. The amino acid sequences of the MoFe α subunit are highly conserved among different phyla (18). The V75I substitution in the suspected gas channel of NifD2 of Anabaena variabilis (equivalent to V70 in A. vinelandii) resulted in greatly diminished N₂ fixation, while allowing for H₂ production rates (under N₂) that were similar to those of wild-type cells under Ar (55). Significantly, however, the nonheterocyst nitrogenase of this strain, which is expressed mainly in vegetative cells under anaerobic conditions, is incompatible with O₂-evolving photosynthesis and thus requires continuous anaerobic conditions along with a supply of exogenous reducing sugars for H₂ production. Substitutions of selected amino acids in the vicinity of the FeMo-co active site within Azotobacter vinelandii nitrogenase were shown to eliminate or greatly diminish N₂ fixation while, in some cases, allowing for effective proton reduction (2, 10, 17, 27, 36, 44, 45, 48). Therefore, certain amino acid exchanges near FeMo-co might produce variant MoFe proteins in heterocyst-forming Anabaena that redirect the electron flux through the enzyme preferentially to proton reduction so as to synthesize more H₂ in the presence of N₂ in an aerobic environment.To examine whether Anabaena sp. strain PCC 7120 nitrogenase can be modified to increase photobiological H₂ production by effecting such a redirection, we evaluated in vivo H₂ production and acetylene reduction rates of a series of cyanobacterial nifD site-directed mutants. We mutated six NifD residues (Fig. ​(Fig.1)1) predicted to lie within 5 Å of FeMo-co to create 49 variants using an Anabaena ΔNifΔHup (previously denoted ΔhupL) parental strain that lacks both an intact nifD and an uptake hydrogenase (34). In an atmosphere containing N₂ and O₂, several mutants exhibited significantly enhanced rates of in vivo H₂ production and accumulated high levels of H₂ compared to the reference strains.Open in a separate windowFIG. 1.Side-on (left) and Mo end-on (right) views of the predicted active site for nitrogenase of Anabaena sp. strain PCC 7120. The FeMo-co cluster, a [7Fe-8S-Mo-X-homocitrate] complex, where X is a central unidentified light atom (N, C, or O), and its two coordinating residues (C282 and H449) are shown in a ball-and-stick representation. Water molecules near the FeMo-co are indicated by isolated spheres in red. The side chains of the residues targeted for mutagenesis—Q193, H197, Y236, R284, S285, and F388—are shown in stick representation. Residues V362 through P367 are represented by lines. The Anabaena residues were mapped onto the corresponding residues from the crystal structure of the A. vinelandii enzyme (PDB file 1M1N). The figure was generated by using PyMOL (www.pymol.org/), with the following color scheme: Fe, orange; S, yellow; C, gray; N and central atom X, blue; O, red; and Mo, pink.     

A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography

A systematic method for the sensitive, precise and accurate determination of hair lipids, including trace amounts of intrinsic endogenous cholesterol (CH), ceramide/N-palmitoyl-DL-dihydrosphingosine (CER/PDS), cholesterol sulfate (CS) and chemically bound 18-methyl eicosanoic acid (18-MEA), has been developed in combination with TLC/FID (flame ionization detection), LC/MS and GC/MS. TLC/FID was used for the simultaneous determination of squalene (SQ), wax esters (WEs), triglycerides (TGs) and free fatty acids (FFAs). Optimal conditions for LC/MS to determine CS and 18-MEA were developed using selected ion monitoring (SIM) under the negative ion mode of electrospray ionization. An alternative procedure for the determination of 18-MEA was also established using commercially available heneicosanoic acid (HEA). In GC/MS, the optimal selection of ions for SIM of trimethylsilylated CH and CER/PDS, and the use of on-column injection has enabled their simultaneous detection. This newly developed method has been used to characterize the hair lipid composition from the proximal root end to the distal tip of chemically untreated hair fibers from two different females, and specific changes of hair lipids probably due to its origin and individuals have been demonstrated for the first time. This method may be useful for clarifying the important roles of intrinsic endogenous 18-MEA, CS, CH and CERs in the function of the cell membrane complex of hair fibers.     

Voltage- and time-dependent action of histrionicotoxin on the endplate current of the frog muscle

Histrionicotoxin, a toxin isolated from skin secretions of a Colombian arrow poison frog, Dendrobates histrionicus, decreased the amplitude and time-course of the endplate current, and altered the voltage dependence of the half-decay time. In addition, the toxin produced a characteristic nonlinearity in the current-voltage relationship of the endplate current when 3-s voltage conditioning steps were used. Reduction in time of the conditioning steps to 10 ms made the current-voltage relationship linear. The decrease in peak amplitude of the endplate current (epc) produced by histrionicotoxin measured during long hyperpolarizing conditioning steps was fitted by a single exponential function. The calculated rate constants ranged from 0.03 to 0.14 s-1 and varied with membrane potential at hyperpolarizing levels. The voltage- and time-dependent action of histrionicotoxin does not require an initial activation of receptors by acetylcholine (ACh). The characteristic of the current-voltage relationship can be accounted for by the observed voltage and time dependency of the attenuation of the endplate current amplitude in the presence of histrionicotoxin during long conditioning steps. These effects of histrionicotoxin on the peak amplitude, and on the voltage and time dependence of the epc were concentration-dependent and slowly reversible upon washing out the toxin. Thus, the voltage- and time-dependent action of histrionicotoxin at the endplate is related to an increase in the affinity between the toxin and the ACh receptor-ionic channel complex. This increase in affinity is postulated to be due to a conformational change of the macromolecule in the presence of histrionicotoxin which is demonstrated to be relatively slow, i.e., on the order of tens of seconds.     

Involvement of the K+‐Cl− co‐transporter KCC2 in the sensitization to morphine‐induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti‐anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K⁺‐Cl^? co‐transporter 2 (KCC2) in the sensitization to morphine‐induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ‐aminobutyric acid A‐type receptor (GABA_AR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine‐induced hyperlocomotion, which is accompanied by the up‐regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down‐regulation of protein phosphatase‐1 (PP‐1) as well as the up‐regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP‐1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre‐treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine‐induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ‐PP‐1‐KCC2 pathway by chronic treatment with zolpidem.     

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.     

Uptake and release of 45Ca by Myxicola axoplasm

The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP- dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.     

Flexible plastic bioreactors for photobiological hydrogen production by hydrogenase-deficient cyanobacteria

Uptake hydrogenase mutant cells of the cyanobacterium Nostoc sp. PCC 7422 photobiologically produced H(2) catalyzed by nitrogenase for several days in H(2)-barrier transparent plastic bags, and accumulated H(2) in the presence of O(2) evolved by photosynthesis. Their H(2) production activity was higher in the sealed flexible bags than in stoppered serum bottles of fixed gas volume.