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Brian F. Thomas A. Robert Jeffcoat Mary W. Myers James M. Mathews C. Edgar Cook 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1994,655(2)
A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C18), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique. 相似文献
3.
The Halide ion-catalysed reaction of benzyl exo-2,3-O-benzylidene-α-
-rhamnopyranoside with tetra-O-benzyl-α-
-galactopyranosyl bromide and hydrogenolysis of the exo-benzylidene group of the product 2 gave benzyl 3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-
-galactopyranosyl)-α-
-rhamnopyranoside (6). Compound 2 was converted into 4-O-α-
-galactopyranosyl-
-rhamnose. The reaction of 6 with tetra-O-acetyl-α-
-glucopyranosyl bromide and removal of the protecting groups from the product gave 4-O-α-
-galactopyranosyl-2-O-β-
-glucopyranosyl-
-rhamnose. 相似文献
4.
Lucjan J. J. Hronowski Walter A. Szarek George W. Hay Anita Krebs William T. Depew 《Carbohydrate research》1989,190(2):203-218
The synthesis of 1-O-β-lactosyl-(R,S)-glycerols was achieved by three methods: (a) in 25% yield by the trimethylsilyl trifluoromethanesulfonate-promoted reaction of octa-O-acetyl-β-lactose (11) with 0.5 mol-equiv. of 2-O-benzylglycerol (4), (b) in 34% yield by the coupling of 4 with an equimolar amount of hepta-O-acetyl--lactosyl bromide (12) in the presence of mercury(II) cyanide, and (c) in 50% yield by the coupling of equimolar amounts of 12 and 1,2-di-O-benzyl-(R,S)-glycerols in the presence of mercury(II) cyanide, followed in each case by the removal of the blocking groups. 1,3-Di-O-β-lactosylglycerols were prepared in 21% yield by the coupling of 11 with 0.5 mol-equivalent of 4 by method (a), and in 38% yield by the coupling of 12 with 0.5 mol-equiv. of 4 by method (b), followed by the removal of the blocking groups. The configuration of the glycosidic linkage between the lactose units and the glycerol residue was established by high-resolution, two-dimensional 1H-n.m.r. spectroscopy. 相似文献
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Carmen S. M. Yong Janelle Sharkey Belinda Duscio Ben Venville Wei-Zen Wei Richard F. Jones Clare Y. Slaney Gisela Mir Arnau Anthony T. Papenfuss Jan Schr?der Phillip K. Darcy Michael H. Kershaw 《PloS one》2015,10(9)
The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes. 相似文献
6.
TLCK (N-α-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50= 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-α-tosyl-L-phenylalanine chloromethyl ketone) (IC50=8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.Protein kinase CTumor promotionProtease inhibitor 相似文献
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Escherichia coli β-D -galactosidase (E.C. 3.2.1.23) was immobilized in cellulose nitrate membrane microcapsules and the reaction kinetics with o-nitrophenyl-β-D -galactopyranoside (ONPG), lactose, and whole milk were studied using both continuous stirred tank and packed bedreactor configurations. The results of the experiments gave effectiveness factors of 0.3 for ONPG, 0.6 to 0.7 for lactose in solution, andclose to unity for lactose in milk. Using a coupled mass transfer and kinetic model, it was possible to estimate the permeability of the microcapsule membrane from the reactor data. Membrane permeabilities on the order of 5 × 10?3 and 3 × 10?4 cm/sec were estimated for ONPG and lactose, respectively. It was determined that the membrane was the limiting mass transfer resistance for the overallreaction. The analysis showed that within the microcapsule, the reaction is reaction rate limited for lactose and slightly diffusion limitedfor ONPG. 相似文献
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Yingsi Chen Ming Jin Tobore Egborge George Coppola Jamie Andre David H. Calhoun 《Protein expression and purification》2000,20(3):472
Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme α-galactosidase A. This enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. An improved method of production of recombinant α-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human α-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human α-galactosidase A. Recombinant human α-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25°C, but not at the standard growth temperature of 30°C. The recombinant α-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant α-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates. 相似文献
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Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice
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Denise E. Sabatino Amanda P. Cline Patrick G. Gallagher Lisa J. Garrett George Stamatoyannopoulos Bernard G. Forget David M. Bodine 《Molecular and cellular biology》1998,18(11):6634-6640
During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells. 相似文献
13.
人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文) 总被引:1,自引:0,他引:1
通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。 相似文献
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乙型肝炎病毒3’末端缺失的preS/S基因在转基因小鼠中的表达 总被引:1,自引:1,他引:0
从乙型肝炎病毒 adr亚型的基因组 DNA中分离 3’末端缺失的preS/S基因的 DNA片段,构建了由CMV启动子控制的真核表达载体。采用受精卵显微注射方法,获得了基因组整合有3’末端缺失的preS/S基因的2个转基因小鼠品系。在不同的时间点采取血清进行了ELISA分析,发现在这2个小鼠品系中3’末端缺失的preS/S基因可被表达,而且呈稳定状态。此小鼠品系的建立,对于探讨乙型肝炎病毒3’末端缺失的preS/S基因的表达产物在体内的生物学作用及其与肝细胞内细胞癌基因的转录激活之间的关系有重要意义。 相似文献
15.
Paul Nugent Merle Potchinsky Cynthia Lafferty Robert M. Greene 《Experimental cell research》1995,220(2)
We have previously shown that both transforming growth factor-β (TGF-β) and retinoic acid (HA) regulate the expression of cellular retinoic acid binding proteins (CRABP) I and II and TGF-β3 mRNAs in primary cultures of murine embryonic palate mesenchyreal (MEPM) cells. We now describe additional crosstalk between the RA and TGF-β signal transduction pathways—the ability of TGF-β, including the endogenous form(s), to modulate the expression of the nuclear retinoic acid receptor-β (RAR-β). Northern blot hybridization revealed that RA induced the expression of RAR-β mRNA, there being little or no detectable expression in untreated MEPM cells. Induction by 3.3 μM RA was abrogated by simultaneous treatment with TGF-β1 (5 ng/ml). TGF-β1 alone had no effect on RAR. mRNA expression. Determination of RAR-β mRNA half-life by treatment with actinomycin D indicated that TGF-β1 did not alter the stability of RAR-β mRNA. Conditioned medium (CM) from MEPM cells contained little active TGF-β protein; heat treatment of the CM dramatically increased the amount of active TGF-β as assessed by the mink lung epithelial cell bioassay. Furthermore, heat- or acid-activated CM also inhibited CRABP-I and RA-induced RAR-β expression. The effect of heat-activated conditioned medium could be abrogated with panspecific neutralizing antibodies to TGF-β, confirming that endogenous TGF-β is the biologically active factor in heat-activated CM. These results provide evidence for complex interactions between TGF-β and RA in the regulation of gene expression in embryonic palatal cells and suggest a role for endogenous TGF-β in the regulation of expression of genes encoding elements of the RA signal transduction pathway. 相似文献
16.
The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheepK15gene, focusing on its expression in the follicles of sheep and mice. We show thatK15is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells areK15-negative. In the follicle bulbK15is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere,K15is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheepK15expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheepK15gene construct exhibited faithful expression and showed no phenotypic consequences ofK15overexpression. An investigation of transgene expression showed thatK15is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle,K15expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like. 相似文献
17.
Michael C. Koss 《Prostaglandins & other lipid mediators》1976,12(6):997-1004
Prostaglandin F2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F1α, F2β and F1β also cause the same cardiovascular effects as F2α, there is a decrease in potency for all parameters measured, with PGF2α>PGF1α>PGF2β>PGF1β. When compared to the actions of PGF2α in producing an increase in pulmonary arterial pressure, PGs F1α, F2β and F1β were less potent by approximately 10, 100, and 1000 fold respectively. 相似文献
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Vince Pozsgay 《Carbohydrate research》1981,90(2)
O-α-
-Rhamnopyranosyl-(1→3)-
-rhamnopyranose (19) and O-α-
-rhamnopyranosyl-(1→2)-
-rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α-
-rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α-
-rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β-
-galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α-
-rhamnopyranosyl)-α-
-rhamnopyranosyl]-β-
-galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α-
-rhamnopyranosyl)-β-
-rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β-
-galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α-
-rhamnopyranosylation at HO-2 or -3 of a free α-
-rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α-
-Rhamnopyranosylation of HO-6 of gb-
-galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O2,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom. 相似文献