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81.
82.
83.
O W Petersen P E H?yer J Hilgers P Briand B van Deurs 《Virchows Archiv. B, Cell pathology including molecular pathology》1985,50(1):27-42
Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined. 相似文献
84.
Ratul Saha Robert S. Donofrio Susan T. Bagley 《Journal of industrial microbiology & biotechnology》2010,37(8):843-848
A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of
three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms
of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since
they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but
are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both
whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 101 colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining
concentrations from used MWF samples, indicating levels between 2.9 × 103 and 3.9 × 106 CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 101 to 1.4 × 105 CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently
used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs. 相似文献
85.
Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat 总被引:1,自引:0,他引:1
The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane. 相似文献
86.
M. I. Jsselstijn J. Kaiser F. L. van Delft H. E. Schoemaker F. P. J. T. Rutjes 《Amino acids》2003,24(3):263-266
Summary. Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed.
The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed
ring-closing alkyne metathesis reactions.
Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002
Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial
aid from the Netherlands Technology Foundation (STW).
Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen,
The Netherlands, E-mail: rutjes@sci.kun.nl
2, selected data: 1H NMR (300 MHz, CDCl3) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.
Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat
gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH2) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature,
trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane
(69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc.
CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition
of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent
was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was
extracted using heptane, washed with brine, dried (MgSO4) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; 1H NMR (300 MHz, CDCl3) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C17H27NO4 309.1940, found 309.1937.
A solution of the tungsten catalyst (7 mg, 10 mol%) in C6H5Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C6H5Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash
column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]D =–14.6 (c = 1, CH2Cl2); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; 1H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for
C18H28N2O5 相似文献
87.
A. Verrips Gerry C. H. Steenbergen-Spanjers J. A. F. M. Luyten R. A. Wevers John H. J. Wokke Fons J. M. Gabreëls Bert G. Wolthers Lambert P. W. J. van den Heuvel 《Human genetics》1997,100(2):284-286
We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis:
a G→A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66
amino acids in the CYP 27 enzyme molecule.
Received: 15 March 1997 / Accepted: 26 March 1997 相似文献
88.
89.
M J Blommers H J Slot G A van der Marel J H van Boom C W Hilbers 《Journal of biomolecular structure & dynamics》1990,8(2):233-251
The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides. 相似文献
90.
The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence 总被引:3,自引:0,他引:3
I R van Driel P A Stearne B Grego R J Simpson J W Goding 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(6):3220-3224
The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined. 相似文献