Measurement of steroid synthesis in zona glomerulosa cells by liquid chromatography-electrospray ionization-mass spectrometry: inhibition by nitric oxide

A liquid chromatography-electrospray ionization-mass spectrometry method was developed to simultaneously determine the concentrations of aldosterone, corticosterone, cortisol, deoxycorticosterone, pregnenolone, and progesterone in bovine adrenal zona glomerulosa (ZG) cells. Steroids were extracted by liquid-liquid extraction, separated on a reverse-phase C18 column, ionized by electrospray, and detected by single-quadrupole mass spectrometry in a positive ion mode. All steroids formed sodium adducts at high abundance. Factors affecting the formation and signal of sodium adducts were investigated. The limits of detection (S/N=3) using selected ion monitoring are 2 pg for these steroids and 10 pg for pregnenolone. DETA NONOate, a nitric oxide donor, inhibited the basal, angiotensin-II-stimulated, and 25-hydroxycholesterol-stimulated syntheses of these steroids in ZG cells in a concentration-dependent manner. The technique demonstrates the ability to determine the individual steroid in each enzymatic step of aldosterone synthesis and the activity of steroidogenic enzymes in adrenal ZG cells.     

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.     

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.     

黄酮化合物色谱保留时间与其三维结构的关系研究

利用比较分子相似性指数分析（CoMSIA）方法,结合黄酮类化合物含有较多羟基、易形成较强分子内氢键的特点,建立了黄酮类化合物色谱保留时间与其三维结构的关系模型,以探讨黄酮类化合物色谱保留时间预测的新方法。模型交叉验证相关系数q2值为0．705,非交叉验证相关系数r2为0．981,表明模型具有较好的预测能力。该研究结果对进一步开展黄酮类化合物液相色谱保留参数与三维结构关系的研究提供了思路和方法。     

Resource allocation among worker castes of the leaf-cutting ants Acromyrmex subterraneus subterraneus through trophallaxis

The division of labor between the different worker castes of leaf-cutting ants may reflect in their capacity to exchange liquids by trophallaxis. The crop capacity of and trophallactic exchanges between different size classes of worker leaf-cutting ants of the sub-species Acromyrmex subterraneus subterraneus were investigated. Size classes were defined from head capsule widths and crop capacity of each class was determined following ad libitum feeding on dye solution. Experiments were carried out to investigate trophallactic exchanges between donor ants and recipient ants of each class size combination on a one to one basis. An experiment was also performed to investigate dye distribution within mini-colonies following introduction of three classes of donor ants. Worker ants were categorized into four size classes from their head capsule widths (C1 = 0.8-1.0 mm; C2 = 1.2-1.5 mm; C3 = 1.6-2.0 mm; C4 = 2.1-2.4 mm). C1 ants crop capacity was 0.13 μL; C2: 0.21 μL; C3: 0.52 μL; C4: 1.03 μL. Ants of each class previously fed on the dye solution (donors) were placed individually with an unfed ant of each class (recipients) and the presence of dye solution, passed from the donor to the recipient by oral trophallaxis was observed after 1 h. Results showed that all classes of donor ants performed trophallactic exchanges with all recipient classes. However, statistically fewer exchanges were seen for C2 donor ants when placed with C3 recipient ants. Ten donor ants of each of three classes (C2, C3 and C4) were introduced into mini-colonies without queen ants. It was observed that C1 and C2 ants were poor recipients, whilst C3 and C4 received the highest percentages of dye. Within 10 h of introducing the donor ants, 14 to 20% of their nest-mates had received dye solution, with 58 to 77% of dye passed to recipients. These studies show the altruistic nature of “food-laden” leaf-cutters and indicate that ants involved in garden maintenance activity are less likely to receive liquids from foraging workers.     

Eye lens proteomics

Summary. The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.     

Prediction of charge-induced molecular alignment: residual dipolar couplings at pH 3 and alignment in surfactant liquid crystalline phases

Recently we reported that the alignment tensor of a biological macromolecule, which was dissolved in a dilute suspension of highly negatively charged filamentous phage at close to neutral pH, can be predicted from the molecule’s 3D charge distribution and shape (Zweckstetter et al. 2004). Here it is demonstrated that this approach is also applicable to alignment of proteins in liquid crystalline phases formed by filamentous phage at low pH. Residual dipolar couplings (RDCs) predicted by our simple electrostatic model for the B1 domain of protein G in fd phage at pH 3 fit very well with the experimental values. The sign of charge–shape predicted one-bond ¹H–¹⁵N dipolar couplings for the B1 domain of protein G (GB1) was inverted at pH 3 compared to neutral pH, in agreement with experimental observations. Our predictions indicate that this is a feature specific for GB1. In addition, it is shown that RDCs induced in the protein ubiquitin by the presence of a positively charged surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide can be predicted accurately by a simple electrostatic alignment model. This shows that steric and electrostatic interactions dominate weak alignment of biomolecules for a wide range of pH values both in filamentous phage and in surfactant liquid crystalline phases.     

Cryovial with partial membrane sealing can prevent liquid nitrogen penetration in submerged storage

Cryopreservation is one of the fundamental techniques in life science. To preserve the viability of cells and tissues, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the non-sterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. To prevent these drawbacks while retaining the benefit of constant ultra-low temperature in submerged liquid nitrogen, we used a heat-sealable membrane to cover the upper portion of vials. After heat-sealing, the vials were completely free of liquid nitrogen penetration in the submerging test. Moreover, the sealing process did not affect the cell viability. This modified protocol provides an easy and efficient tool to ensure the integrity of biospecimens in long-term storage without interfering with existing cryobox storage systems.     

Basidiomycete cryopreservation on perlite: evaluation of a new method

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.     

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.