Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C₈ cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20–1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.     

Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

High resolution scanning electron microscopy of plant chromosomes

A preparation technique for high resolution field emission scanning electron microscopy of plant chromosomes is described. The technique was optimized to use standard squash preparations of mitotic and meiotic chromosomes from root tips of barley, wheat, and rye. After light microscopic observation and documentation, the same object can be investigated with a 100-fold higher resolution using a field emission scanning electron microscope. Tilting of the specimens provides a three-dimensional insight into chromosomal structures at different stages of condensation and decondensation. With this technique it was possible to document for the first time at high resolution structures such as the centromeric region, the spindle apparatus and the spindle fibre attachment region. The smallest unit of the DNA packed that can be resolved is a beads on a string-like structure of the nucleofilament (10–15 nm fibre).by D. Schweizer     

Preparation of phosphopeptide thioesters by Fmoc- and Fmoc(2-F)-solid phase synthesis

Summary Efficient methods for the preparation of phosphopeptide thioesters were examined, using Fmoc-based solid-phase method. Phosphopeptide thioesters were obtained in good yields by the use of 1-methylpyrrolidine hexamethyl-eneimine and 1-hydroxybenzotriazole in a DMSO-DMF (1∶1, v/v) solution for deblocking the Fmoc groups. Epimerization, which is often observed at the C-terminal amino acid, was effectively suppressed by shortening the time of deblocking process via the use of highly base sensitive Fmoc(2-F) groups for α-amino protection.     

Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation

A procedure for preparation of purified cellodextrins in gram quantities was developed for use in biochemical and microbiological studies. Cellodextrins were prepared by hydrolyzing microcrystalline cellulose (Avicel) over a period of 4 to 5.5h in the presence of a mixture of 80% (v/v) concentrated hydrochloric acid ( approximately 37 wt.%) and 20% (v/v) concentrated sulfuric acid ( approximately 98 wt.%) at room temperature (22 degrees C). Acetone precipitation, washing ion exchange, and neutralization with barium hydroxide were used to generate a solution of mixed cellodextrins substantially free of acids and salts. Yields following hydrolysis and precipitation were approximately 0.05, approximately 0.07, approximately 0.06, and approximately 0.02 g/g cellulose for cellotriose (G(3)), cellotetraose (G(4)), cellopentose (G(5)), and cellohexose (G(6)), respectively. Cellodextrins with degrees of polymerization from 3 to 11 were separated chromatographically using a 29 x 5-cm I.D. Bio-Rad AG50W-X4 column arranged in series with a 91 x 5-cm I.D. Bio-Gel P4 column. This two-column system was used to obtain cellodextrin preparations at 240 mg/day for G(3), 330 mg/day for G(4), 260 mg/day for G(5), and 130 mg/day for G(6), with purity >99% for G(3), G(4), and G(5) and >95% for G(6). The overall procedure achieves yields comparable to the highest previously reported, employs a separation system that can readily be reused for multiple runs, and avoids use of fuming HCl.     

Ion-exchange chromatography in the presence of the non-ionic dissociating agent chloral hydrate. Application to a membrane protein, bovine heart cytochrome c oxidase.

In previous work we have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) is a potent non-ionic protein dissociating agent. We have employed it in systems of polyacrylamide-gel electrophoresis and have demonstrated the presence of 15 components in a preparation of bovine heart cytochrome c oxidase [Griffin & Landon (1981) Biochem. J. 197, 333-344]. Here we describe the use of solutions containing aq. 100% (w/v) chloral hydrate in the ion-exchange column chromatographic separation on CM-cellulose of the alpha- and beta-chains of human haemoglobin, which we have employed as a model protein of known structure. We also describe the use of similar procedures in order to fractionate the polypeptide components of bovine heart cytochrome c oxidase. An effective separation has been obtained and we suggest that chloral hydrate-containing solutions could have general application in the ion-exchange-chromatographic analysis of membrane proteins, a procedure that has had restricted use owing to the inadequacy of non-ionic dissociating agents available previously.     

The laser in the Lowry technique for microdissection of freeze-dried tissue slices

Synopsis A new technique for tissue microdissection is described. This procedure, using an u.v.-laser micropreparation instrument, overcomes the extremely timeconsuming manual preparation. The u.v.-laser micropreparation design allows fast, precise, reproducible and smear-contamination-free tissue microdissection. The preparation of the tissue sample can be programmed by tracing out the area to be sampled with a non-destructive 0.5 m W He-Ne-laser aiming-beam. The trace is stored in a small electronic unit, which then guides the motor-driven stepping stage on the microscope in the actual dissection run with the u.v.-laser.The laser power is adjustable in the range 4 to 40 kW and controlled by a photo diode displayed on an oscilloscope screen. In the tissue slice, prepared according to Lowry, an unlimited number of cells or tissue compartments can be dissected and afterwards weighed. The procedure described offers a broader use of the quantitative microhistochemical techniques of Lowry and of Neuhoff.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September, 1975.     

Determination of telithromycin in human plasma and microdialysates by high-performance liquid chromatography

A high-performance liquid chromatography method for the quantitative determination of telithromycin in biological fluids is described. The method is suitable for plasma and microdialysates from the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue. Plasma samples were deproteinised with trichloroacetic acid and neutralised with sodium hydroxide. Microdialysates were analysed without further preparation step. Telithromycin was separated isocratically on a reverse-phase column using acetonitrile-0.03 M ammonium acetate, pH 5.2 (43:57, v/v) at a flow rate of 0.8 mlmin(-1), and fluorescence detection (excitation 263 nm, emission 460 nm). The calibration curve was linear from 0.01 to 5 microgml(-1). Within- and between-day imprecision and inaccuracy was < or =10%. The limits of quantification were 0.02 and 0.015 microgml(-1) for plasma and microdialysates, respectively. Since telithromycin is decomposed in aqueous solution at ambient temperature, it is strongly recommended to store samples frozen at -80 degrees C, to maintain the temperature at 4 degrees C during all preparation steps, and to analyse samples within 120 min after thawing.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.     

Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

切割延髓、酸碱度及温度对新生大鼠离体延髓—脊髓标本呼吸节律性放电的影响

目的和方法：在新生大鼠离体延髓－－脊髓标本上,用吸附电极记录膈神经根－颈4、颈5腹根（C4v,C5v)和舌下神经根（Ⅻ）节律性放电活动(rhythmical discharge activity,RDA),并观察其在对延髓微切割、改变灌流液pH值及温度后RDA的变化。结果：①新生鼠离体延髓－脊髓标本在这灌流条件下,可存活6－8h,持续4h可稳定记录到C4v、C5v和Ⅻ的RDA,且两者完全同步,为呼吸节律性放电（RDA,RRDA）;②从延髓头端向尾端切割（每次100μm）,切至闩前500μm水平时,RRDA不变,进一步切割,至闩水平,RRDA消失;③从尾端向头端切割,切至舌下神经根下缘水平,RRDA不变,进一步向头端切割,RRDA逐渐消失;④从延髓背侧向腹侧水平切割,切割至背→腹一半水平时,RRDA不变,进一步向腹侧切割,RRDA逐渐消失;⑤灌流液的pH值从7.35降至6.85,RRDA逐渐增强,而从6.85降至4.5时,逐渐减弱至消失。pH值从7.45上升到7.85,呼吸频率逐渐减漫,放电幅度增强;⑥温度由27℃上升到37℃,RRDA逐渐增强;由38℃上升到41℃,RRDA逐渐减弱,温度则27℃降23℃,RRDA逐渐减弱。温度为42℃或22℃时,RRDA消失。结论：面神经后核内侧区可能是呼吸节律起源的部位,且此区域的神经元对温度及酸碱度变化敏感。     

Determination of intracellular nitrate.

A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans.     

Determination of the lactone and lactone plus carboxylate forms of 9-aminocamptothecin in human plasma by sensitive high-performance liquid chromatography with fluorescence detection

Two sensitive reversed-phase high-performance liquid chromatographic fluorescence methods, with simple sample handling at the site of the patient, are described for the determination of the lactone and lactone plus carboxylate forms of 9-aminocamptothecin (9AC). For 9AC lactone, the sample preparation was a liquid–liquid extraction with acetonitrile–n-butyl chloride (1:4, v/v), whereas the sample preparation for 9AC total (lactone plus carboxylate) was a simple deproteinization with 5% perchloric acid–methanol (1:1, v/v), which results in the conversion of the carboxylate into the lactone form. The lower limits of quantitation were 50 pg/ml and 100 pg/ml for 9AC lactone and 9AC total, respectively. The within-run precisions at four tested concentrations were ≤6.3% for 9AC lactone and ≤5.3% for 9AC total. The between-run precisions were ≤8.9% and ≤5.6%, respectively. The assays were developed to enable pharmacological analysis of 9AC in a bioavailability and oral phase I study in patients with solid tumors.     

The determination of purine levels in human and mouse plasma

Variable levels of acetic anhydride have been recommended for addition to one of two reagents used in the glyoxylic acid method for the determination of tryptophan. For use of this reagent immediately after preparation it was shown that a minimum of 16% (v/v) of acetic anhydride should be included in the formulation to obtain near-maximum sensitivity. It was further demonstrated that reagent formulations with and without acetic anhydride changed with exposure to light. The observed changes are manifest as changes in the relative sensitivities of the assay. Several modifications are recommended to improve the sensitivity and stability of the acetic anhydride-containing reagent in this assay.     

Modification of the Oligidic Medium for Axenic Culture of Aphelenchoides rutgersi

Aphelenchoides rutgersi was axenically cultured in modified Soytone, yeast extract, lyophilized chick embryo extract medium (3% ST:2% YE:20% CEE-L, w/v:w/v:v/v). Earlier formulations used 10% CEE, v/v, before the manufacturer changed the preparation. After reestablishing A. rutgersi in medium that permitted continuous subcultivadon and reproduction, a second medium was tested that contained 0.5% sucrose and 0.5% Lipid Concentrate. The commercially available Lipid Concentrate made it possible to incorporate nonaqueous soluble chemicals into the medium. In addition, 0.1% Fast Green #3 was added to both media to visually demonstrate active ingestion of nutriment.     

Rapid detection of a fungal Poly (3-hydroxybutyrate) depolymerase using a new in situ gel activity staining technique

A modified and rapid technique has been developed for activity staining of a fungal poly(3-hydroxybutyrate) depoly-merase by incorporating poly(3-hydroxybutyrate) (2.5% w/v) in the polyacrylamide gel under polymerisation and incubating in 100 mM Tris/HCl (pH 8.0) containing 1 mM CaCl at 30°C. This decreased the time required for detection to 4 h from the conventional 12–18 h using solid-plate method. The enzyme preparation failed to penetrate the gel on conventional electrophoresis but treatment with 0.5% (v/v) Triton X-100 facilitated enzyme migration into the gel without its activity being affected.     

Long-term continuous production of optically active 2-(4-chlorophenoxy)propanoic acid by yeast lipase in an organic solvent system

Summary Long-term continuous optical resolution of 2-(4-chlorophenoxy)propanoic acid was carried out by stereoselective esterification with Celite-adsorbed lipase OF 360 from Candida cylindracea using n-tetradecanol as the second substrate in organic solvent systems. The water content of the Celite-adsorbed lipase affected productivity, 1.0 l water·mg lipase^–1 being optimal for preparation of the adsorbed lipase. Water-saturated carbon tetrachloride-isooctane (8:2, v/v) was found to be an excellent organic solvent for the continuous operation. The particle size of Celite had no effect on productivity. Under optimized conditions, the (R)-enantiomer of the acid was continuously esterified with high stereoselectivity in a packed-bed column reactor for 34 days. Furthermore, it was found that treatment of the reactor with acetone made it possible to restore productivity and extend the period of continuous operation for further 29 days. Offprint requests to: A. Tanaka     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.