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1.
Bruce R. Dorrbecker Susan H. Mercik Paul A. Kramer 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1984,336(1)
A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy. 相似文献
2.
Holly A. Ingraham Mehran Goulian 《Biochemical and biophysical research communications》1982,109(3):746-752
Deoxyuridine triphosphatase has been purified from cultured human lymphoid cells in high yield and stable form by a relatively simple procedure. The properties differed somewhat from those reported previously, e.g. apparent Km, molecular weight, and effects of divalent metals. No other naturally occurring dNTP or NTP serves as substrate, however, the enzyme may be an important site of interaction with intracellular derivatives of analogues of dUrd. It is shown here that deoxyuridine triphosphatase acts on araUTP, 6-azadUTP, 2′-FdUTP, and 2′,3′-dideoxyUTP, but the enzyme has no effect on 5-C1dUTP, 5-BrdUTP, 5-HgdUTP and dUrd-5′[α-thio]triphosphate. For the preparation of one of the analogues, the enzyme, trans-N-deoxyribosylase, from Lactobacillus, was used to prepare the deoxynucleoside from the base, a procedure that may have general usefulness. 相似文献
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Jonathan T. Sims Sourik S. Ganguly Holly Bennett J. Woodrow Friend Jessica Tepe Rina Plattner 《PloS one》2013,8(1)
Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis. 相似文献
5.
Dielectric spectroscopy is a non-invasive contact technique that permits the in vivo measurement of the specific electrical properties of biological tissue induced by an external electrical field. Permittivity, relaxation time and specific conductivity as a function of corneal hydration (wet weight/dry weight) and temperature were measured in 10 porcine corneas. Variation of tissue hydration has a minor influence on the signal, with a significant variation of the signal being detectable only for relatively dry tissue. A much greater influence was found for temperature, in particular on relaxation times. Dielectric spectroscopy provides us with an opportunity to detect structural, in particular temperature-induced, changes in living tissue. In the frequency range investigated, hydration has only a small influence on the dielectric properties of the tissue. 相似文献
6.
Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure 总被引:2,自引:0,他引:2
Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA. 相似文献
7.
8.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein kinase activity in murine B cells 总被引:1,自引:0,他引:1
C M Kramer K W Johnson R K Dooley M P Holsapple 《Biochemical and biophysical research communications》1987,145(1):25-33
Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase. 相似文献
9.
The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm 总被引:1,自引:0,他引:1
To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells. 相似文献
10.
Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. 总被引:67,自引:9,他引:58
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P Stanssens C Opsomer Y M McKeown W Kramer M Zabeau H J Fritz 《Nucleic acids research》1989,17(12):4441-4454
An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model. 相似文献