Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement

Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.     

Complex between α-Chymotrypsin and poly(ethylene glycol) catalytically active in organic media

-Chymotrypsin and poly(ethylene glycol) were mixed in an aqueous phosphate buffer solution and lyophilized. The preparation was readily soluble in anhydrous isooctane. The polymer/enzyme complex, i.e., poly(ethylene glycol)/-chymotrypsin, was catalytically active in anhydrous isooctane even when the molecular ratio of them was unity.     

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

Large-scale methacrylate monolithic columns: design and properties

Monoliths represent a special class of chromatographic supports. In contrast to other stationary phases, they consist of a single piece of highly porous material through which a sample is mainly transported by convection. As a consequence, monoliths enable fast separations and exhibit flow-unaffected properties, which make them attractive for purification of macromolecules like proteins or DNA. In this work, methacrylate-based monolithic columns with the bed volume up to 8000 ml are characterized. They perform high-resolution separations of several hundreds of grams of proteins per hour by utilizing liter per minute flow rates. They are incompressible under these operating conditions and resistant to strong alkaline conditions.     

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20-22 hr with 12-15 x 10(6) sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P < 0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P > 0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P > 0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions.     

Tracking the acetate threshold using DO-transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media

DO-transient nutrient controllers use the dissolved oxygen signal to attempt acetate threshold tracking during fed-batch cultivation of recombinant E. coli. Here we apply DO-transient control to the production of Jembrana disease virus protein in complex Super Luria medium and compare performance against a high-limit pH-stat controller. For induction at medium cell density (harvest between 31 and 32.5 g dcw L) a total productivity of 0.27 g L h was achieved as compared to 0.24 g L h with the high-limit pH-stat. For induction at high cell density (harvest at 60 g dcw L), decreased productivity (0.12 g L h) was attributed to the effect of acetate accumulation on recombinant protein formation and a concomitant lowering of the critical growth rate. Our results suggest that complex media provides a difficult environment for the application of acetate threshold tracking DO-transient control because of difficulties in re-oxidizing acetate, and apparent localized production of acetate below the production threshold (as detected by the DO-transient controller as SPOUR(crit)). Configuring the DO-transient controller to avoid aggressive threshold probing is suggested as a means to improve performance and reduce acetate accumulation in complex media.     

Indirect photometric ion chromatographic analysis of anions in Haematococcus pluvialis culture media

An indirect photometric ion chromatographic method for the simultaneous determination of chloride, nitrate and sulfate ions was developed and applied to the determination of anions, mainly nitrate, in the alga Haematococcus pluvialis culture media. Using phthalic acid/sodium tetraborate aqueous solution as the mobile phase, anions can be detected indirectly by a UV detector. The calibration curves for these anions gave good linearity from 1 to 1000 g ml^–1.     

A method for obtaining equilibrium tautomeric mixtures of reducing sugars via glycosylamines using nonaqueous media

Equilibrium tautomeric mixtures of several mono- and disaccharides are obtained in anhydrous form, without the use of water, by reacting the commercially available reducing sugars with ammonia gas in dry methanol, followed by the concentration of the resultant solution to dryness. Mutarotation and hydrolysis of the initially formed glycosylamine in the resultant medium account for the transformation. Equilibrium anomeric mixtures enriched in the beta-form of commercially available sugars such as alpha-D-glucose and alpha-lactose have not only vastly increased solubility, but are also synthetically valuable as these can be readily converted to the methyl/benzyl/trimethylsilyl ether and other derivatives for further transformations.