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101.
Silva MJ Malek NA Hodge CC Reidy JA Kato K Barr DB Needham LL Brock JW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,789(2):393-404
Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC-APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC-APCI-MS/MS using isotope dilution with (13)C(4) internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP. 相似文献
102.
Bagnall W Sharpe PM Newham P Tart J Mott RA Torr VR Forder RA Needham MR 《Protein expression and purification》2003,27(1):1-11
The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs. 相似文献
103.
104.
Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment 总被引:11,自引:2,他引:9
When microbes evolve in a continuous, nutrient-limited environment, natural
selection can be predicted to favor genetic changes that give cells greater
access to limiting substrate. We analyzed a population of baker's yeast
that underwent 450 generations of glucose-limited growth. Relative to the
strain used as the inoculum, the predominant cell type at the end of this
experiment sustains growth at significantly lower steady-state glucose
concentrations and demonstrates markedly enhanced cell yield per mole
glucose, significantly enhanced high-affinity glucose transport, and
greater relative fitness in pairwise competition. These changes are
correlated with increased levels of mRNA hybridizing to probe generated
from the hexose transport locus HXT6. Further analysis of the evolved
strain reveals the existence of multiple tandem duplications involving two
highly similar, high- affinity hexose transport loci, HXT6 and HXT7.
Selection appears to have favored changes that result in the formation of
more than three chimeric genes derived from the upstream promoter of the
HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism
to account for these changes and speculate as to their adaptive
significance in the context of gene duplication as a common response of
microorganisms to nutrient limitation.
相似文献
105.
Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands 总被引:2,自引:2,他引:0
Britten CJ; van den Eijnden DH; McDowell W; Kelly VA; Witham SJ; Edbrooke MR; Bird MI; de Vries T; Smithers N 《Glycobiology》1998,8(4):321-327
The alpha3 fucosyltransferase, FucT-VII, is one of the key
glycosyltransferases involved in the biosynthesis of the sialyl Lewis X
(sLex) antigen on human leukocytes. The sialyl Lewis X antigen
(NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential
component of the recruitment of leukocytes to sites of inflammation,
mediating the primary interaction between circulating leukocytes and
activated endothelium. In order to characterize the enzymatic properties of
the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been
expressed in Trichoplusia ni insect cells. The enzyme is capable of
synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from
3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels
of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies
using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors
demonstrate that FucT-VII is able to synthesize both di-fucosylated and
tri-fucosylated structures from mono- fucosylated precursors, but
preferentially fucosylates the distal GlcNAc within a polylactosamine
chain. Furthermore, the rate of fucosylation of the internal GlcNAc
residues is reduced once fucose has been added to the distal GlcNAc. These
results indicate that FucT-VII is capable of generating complex selectin
ligands, in vitro , however the order of fucose addition to the lactosamine
chain affects the rate of selectin ligand synthesis.
相似文献
106.
R M Hochmuth H P Ting-Beall B B Beaty D Needham R Tran-Son-Tay 《Biophysical journal》1993,64(5):1596-1601
At issue is the type of constitutive equation that can be used to describe all possible types of deformation of the neutrophil. Here a neutrophil undergoing small deformations is studied by aspirating it into a glass pipet with a diameter that is only slightly smaller than the diameter of the spherically shaped cell. After being held in the pipet for at least seven seconds, the cell is rapidly expelled and allowed to recover its undeformed, spherical shape. The recovery takes approximately 15 s. An analysis of the recovery process that treats the cell as a simple Newtonian liquid drop with a constant cortical (surface) tension gives a value of 3.3 x 10(-5) cm/s for the ratio of the cortical tension to cytoplasmic viscosity. This value is about twice as large as a previously published value obtained with the same model from studies of large deformations of neutrophils. This discrepancy indicates that the cytoplasmic viscosity decreases as the amount of deformation decreases. An extrapolated value for the cytoplasmic viscosity at zero deformation is approximately 600 poise when a value for the cortical tension of 0.024 dyn/cm is assumed. Clearly the neutrophil does not behave like a simple Newtonian liquid drop in that small deformations are inherently different from large deformations. More complex models consisting either of two or more fluids or multiple shells must be developed. The complex structure inside the neutrophil is shown in scanning electron micrographs of osmotically burst cells and cells whose membrane has been dissolved away. 相似文献
107.
Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli. 相似文献
108.
BA Payseur HA Covert CJ Vinyard M Dagosto 《American journal of physical anthropology》1999,110(1):115-116
Payseur BA, Covert HA, Vinyard CJ, Dagosto M. 1999. New Body Mass Estimates for Omomys carteri, a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52. This article included an incomplete Table 2. The final two columns, showing “Intercept” and “SEE” data were omitted. The complete Table 2, with these two columns included, is provided below. 相似文献
109.
This article examines the role language ideologies played in the changing instructional and social organization of Khmer literacy classes in Long Beach, California. Language and language use in classrooms have been carefully examined over the years, but analysis of how language attitudes influence pedagogical theory and practice has been largely neglected. This article reveals one way in which language ideologies engage with local theories of learning to shape not only pedagogies informing instruction but also social relations within classes. 相似文献
110.
Bravo R Caltabiano LM Fernandez C Smith KD Gallegos M Whitehead RD Weerasekera G Restrepo P Bishop AM Perez JJ Needham LL Barr DB 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,820(2):229-236
We have developed a method to measure 12 urinary phenolic metabolites of pesticides or related chemicals. The target chemicals for our method are 2-isopropoxyphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; carbofuranphenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; 3,5,6-trichloro-2-pyridinol; para-nitrophenol, ortho-phenylphenol, pentachlorophenol, 1-naphthol and 2-naphthol. The sample preparation involves enzyme hydrolysis, isolation of the target chemicals using solid phase extraction cartridges, a phase-transfer catalyzed derivatization, cleanup using sorbent-immobilized liquid/liquid extraction cartridges, and concentration of the sample. Derivatized samples are analyzed by capillary gas chromatography-tandem mass spectroscopy using isotope dilution calibration for quantification. The limits of detection are in the mid ng/L range and the average coefficient of variation was below 15% for most of the analytes. Using our method, we measured concentrations of the target chemicals in urine samples from the general population. 相似文献