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81.
This report describes a convenient method for the rapid and efficient
release of N-linked oligosaccharides from low microgram amounts of
glycoproteins. A 96-well MultiScreen assay system containing a
polyvinylidene difluoride (PVDF) membrane is employed to immobilize
glycoproteins for subsequent enzymatic deglycosylation. Recombinant
tissue-type plasminogen activator (rt-PA) is used to demonstrate the
deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled
the recovery of a sufficient amount of N-linked oligosaccharides released
enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5
microgram rt-PA for subsequent analysis by matrix-assisted laser
desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The
immobilization of rt-PA to the PVDF membrane did not sterically inhibit the
PNGaseF-mediated release of oligosaccharides from rt-PA as determined by
tryptic mapping experiments. Comparison of the oligosaccharides released
from 50 micrograms of rt-PA by either the 96-well plate method or by a
standard solution digestion procedure showed no significant differences in
the profiles obtained by high-pH anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). Both neutral and sialylated
oligosaccharide standards spiked into wells were recovered equally as
determined by HPAEC-PAD. One advantage of this approach is that reduction
and alkylation can be performed on submicrogram amounts of glycoproteins
with easy removal of reagents prior to PNGaseF digestion. In addition, this
method allows 60 glycoprotein samples to be deglycosylated in 1 day with
MALDI-TOF or HPAEC-PAD analysis being performed on the following day.
相似文献
82.
Strasser-Wozak EM Hartmann BL Geley S Sgonc R Böck G AJ Santos Hattmannstorfer R Wolf H Pavelka M Kofler R 《Cell death and differentiation》1998,5(8):687-693
The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis. 相似文献
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G Petrescu R Verley 《Comptes rendus des séances de la Société de biologie et de ses filiales》1982,176(4):479-482
The neurons which innervate the small common hairs of the mystacial pad are shown to be located in the dorsomedial part of the ganglion of Gasser. In ganglia atrophied as a consequence of early destruction of vibrissae follicles, neurons that innervate these small hairs are still active. 相似文献
86.
Grazziela Y. Nicolau Erhard Haus Mihai Popescu Linda Sackett-Lundeen Elena Petrescu 《Chronobiology international》1991,8(2):149-159
Time of occurrence of cardiac death due to arrhythmia, heart failure, or acute myocardial infarction was recorded in 86 elderly subjects, belonging to a group in whom circadian and circannual rhythms in blood pressure and urinary catecholamine excretion had been studied previously. All patients were retired, with no work responsibilities, and lived-closely-supervised in a home for the aged-on a routine that provided little differences between weekdays and weekends. Cardiac mortality showed a circadian variation, with a peak in the early morning hours, coinciding with the circadian peak in systolic and diastolic blood pressures. A weekly (circaseptan) variation in cardiac mortality was found, with the greatest number of patients dying on Mondays and the least on Thursdays. There were seasonal differences in cardiac mortality, with a peak in July and a broader peak during the cold season (December to February). The former coincides with the circannual peak in diastolic blood pressure, but is unrelated to the seasonal variation in norepinephrine excretion. Circadian, circaseptan, and circannual variations in cardiac mortality appear to be the expression of time-dependent, transient risk states for catastrophic cardiac events, which may lend themselves to preventive treatment. 相似文献
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Stefana Petrescu Stefan A. Hulea Rodica Stan Dorina Avram Victoria Herlea 《Biotechnology letters》1992,14(1):1-6
Summary The bioconversion of D-galacturonic acid to L-ascorbic acid was demonstrated in a new yeast strain isolated from the Japanese Crystal. Both intact cells and a crude mitochondrial extract yielded L-ascorbic acid when D-galacturonic acid was present. 相似文献
90.