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71.
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
72.
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
73.
Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current
and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal
bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell
bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating
current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition
remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were
sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three
potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control
of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the
noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days,
indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally
expressed on the giant axon membrane to the somatic membrane.
Received: 24 October 1995/Revised: 5 March 1996 相似文献
74.
Validation and use of centrifugal ultrafiltration-dialysis in the measurement of percent free oestradiol in serum 总被引:1,自引:0,他引:1
M Dowsett M D Mansfield D J Griggs S L Jeffcoate 《Journal of steroid biochemistry》1984,21(3):343-345
The validity of the centrifugal ultrafiltration-dialysis method in the measurement of the percentage of oestradiol and testosterone which is not bound to protein (free) in human serum was assessed. In an initial validation, the values of % free steroid obtained for oestradiol and testosterone in protein-free ultrafiltrates of human serum were higher than expected. Filter paper discs used in the system were shown to adsorb [3H]oestradiol from protein-free solution. Their replacement with glass-fibre discs, which did not adsorb oestradiol, decreased but did not eliminate the discrepancy between observed and expected results for % free oestradiol in ultrafiltrates. The use of polyethylene rather than polypropylene vials then reduced the discrepancy to insignificant levels. The estimates for % free oestradiol in serum were reduced by these modifications. 相似文献
75.
76.
77.
Using nitrate non-utilizing (nit) mutants, we determined vegetative compatibility groups (VCG) among strains of Beauveria bassiana representing strains indigenous to North America, isolated from diverse insect hosts, and strains that have been mass released for insect control. Genetic similarity among these strains was analyzed using random amplified polymorphic DNA (RAPD) markers. Our data revealed 23 VCGs among the 34 strains tested, with most of these groups comprised of only a single strain. We also observed a VCG comprised of eight genetically similar strains isolated from Colorado potato beetles (CPB). Co-inoculation studies of CPB larvae with complementary nit mutants from the same or from different VCGs revealed heterokaryosis in four out of five same-VCG pairs, with only 5-17% of the sporulating cadavers generating few parasexual recombinants. In contrast, none of the infected beetles treated with non-compatible pairs generated recombinants. The large number of VCGs observed and the low frequency of in vivo recombination limited to vegetatively compatible strains indicate that this self/non-self recognition system may be an effective barrier preventing genetic exchange between dissimilar strains in the field. 相似文献
78.
Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence 总被引:4,自引:1,他引:3
The Alcidae is a unique assemblage of Northern Hemisphere seabirds that
forage by "flying" underwater. Despite obvious affinities among the
species, their evolutionary relationships are unclear. We analyzed
nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b
gene and allelic profiles for 37 allozyme loci in all 22 extant species.
Trees were constructed on independent and combined data sets using maximum
parsimony and distance methods that correct for superimposed changes.
Alternative methods of analysis produced only minor differences in
relationships that were supported strongly by bootstrapping or standard
error tests. Combining sequence and allozyme data into a single analysis
provided the greatest number of relationships receiving strong support.
Addition of published morphological and ecological data did not improve
support for any additional relationship. All analyses grouped species into
six distinct lineages: (1) the dovekie (Alle alle) and auks, (2)
guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets,
(5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and
puffins. The two murres (genus Uria) were sister taxa, and the black
guillemot (Cepphus grylle) was basal to the other guillemots. The Asian
subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was
the most divergent brachyramphine murrelet, and two distinct lineages
occurred within the synthliboramphine murrelets. Cassin's auklet
(Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other
auklets and puffins, respectively, and the Atlantic (Fratercula arctica)
and horned (Fratercula corniculata) puffins were sister taxa. Several
relationships among tribes, among the dovekie and auks, and among the
auklets could not be resolved but resembled "star" phylogenies indicative
of adaptive radiations at different depths within the trees.
相似文献
79.
Carol?Brown Thomas?Griggs Ida?Holaskova Jeff?SkousenEmail authorView authors OrcID profile 《Bioenergy Research》2016,9(1):40-49
Switchgrass (Panicum virgatum L.), a warm-season perennial grass, is an important bioenergy crop candidate because it produces high biomass yields on marginal lands and on reclaimed surface mined sites. In companion studies, dry matter (DM) yields for Cave-in-Rock, Shawnee, and Carthage cultivars varied from 4.2 to 13.0 Mg ha?1averaged over 6 years at the reclaimed Hampshire site, and fertilization increased yields of Cave-in-Rock at Black Castle and Coal Mac sites from 0.3 to 2 Mg ha?1 during the first 3 years. The objective of these experiments was to compare the impacts of cultivar and soil amendments on biomass quality and theoretical ethanol production of switchgrass grown on surface mines with differing soil characteristics. Biomass quality was determined for fiber, ash, lignin, digestibility, and carbohydrate contents via near-infrared reflectance spectroscopy, and carbohydrates were used to calculate theoretical ethanol yield (TEY; L Mg?1) and multiplied by biomass yield to calculate theoretical ethanol production (TEP; L ha?1). Cultivars at the Hampshire site did not differ in TEY and ranged from 426 to 457 L Mg?1. Theoretical ethanol production from Cave-in-Rock at Hampshire was 7350 L ha?1, which was higher than other cultivars because of its greater biomass production. This TEP was higher than in other studies which predicted 4000 to 5000 L ha?1. At the Black Castle and Coal Mac sites, fertilizer applications slightly affected biomass quality of switchgrass and TEY, but provided greater TEP as a function of increased yield. Similar to other findings, total switchgrass biomass production has more impact than compositional differences on TEP, so maximizing biomass production is critical for maximizing potential biofuel production. With appropriate soil substrates, fertilization, planning, and management, large areas of reclaimed surface mines can be converted to switchgrass stands to produce high biomass quality and yields to support a bioethanol industry. 相似文献
80.
Growing food crops for biofuel on productive agricultural lands may become less viable as requirements to feed a growing human population increase. This has increased interest in growing cellulosic biofuel feedstocks on marginal lands. Switchgrass (Panicum virgatum L.), a warm-season perennial, is a viable bioenergy crop candidate because it produces high yields on marginal lands under low fertility conditions. In other studies, switchgrass dry matter (DM) yields on marginal croplands varied from 5.0 to 10.0 Mg ha?1 annually. West Virginia contains immense areas of reclaimed surface mined lands that could support a switchgrass-based biofuel industry, but yield data on these lands are lacking. Field experiments were established in 2008 to determine yields of three switchgrass cultivars on two West Virginia mine sites. One site reclaimed with topsoil and municipal sludge produced biomass yields of 19.0 Mg DM ha?1 for Cave-in-Rock switchgrass after the sixth year, almost double the varieties Shawnee and Carthage, at 10.0 and 5.7 Mg ha?1, respectively. Switchgrass yields on another site with no topsoil were 1.0 Mg ha?1 after the sixth year, with little variation among cultivars. A second experiment was conducted at two other mine sites with a layer of topsoil over gray overburden. Cave-in-Rock was seeded with fertilizer applications of 0, 34, and 68 kg N-P2O5-K2O ha?1. After the third year, the no fertilizer treatment averaged biomass yields of 0.3 Mg ha?1, while responses to the other two rates averaged 1.1 and 2.0 Mg ha?1, respectively. Fertilization significantly increased yields on reclaimed mine soils. Where mine soil fertility was good, yields were similar to those reported on agricultural soils in the Northeastern USA. 相似文献