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101.
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
102.
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
103.
Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin 总被引:1,自引:0,他引:1
Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms. 相似文献
104.
Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa 总被引:1,自引:0,他引:1
The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement. 相似文献
105.
One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates. 相似文献
106.
A method based on simultaneous short-term exposure to 14 CO2 and 15 N2 is described for studying nitrogen fixation and distribution in legumes relative to carbon assimilation and use. Equipment designed to accomodate experiments under natural conditions with very little disturbance of the N2 fixing association is used. It permits continuous measurement and regulation of variables such as air temperature, humidity and CO2 concentration as well as soil aeration. Measurements of distribution and use of assimilates, respiration of nodulated roots, quantitative N2 fixation and the distribution and fate of fixed N as a function of time lead to a precise estimation of C and N budgets for each labelling period. When experiments are done at several phenological stages they give a new insight into the complex C and N interrelations in legume symbiosis.
A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts.
The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations. 相似文献
A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts.
The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations. 相似文献
107.
108.
J K Voglmayr M Mizumachi D W Washington C L Chen C W Bardin 《Biology of reproduction》1990,42(1):81-86
Adult Suffolk rams were immunized four times against the human recombinant inhibin alpha-subunit over a period of 80 days. Blood samples were collected at weekly intervals and serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were determined by radioimmunoassay procedures. The results show that season-related elevations of gonadotropin levels in immunized rams was delayed by 1-2 wk and, in these animals, it was more pronounced and extended than in vehicle-treated controls. Peaks of circulating testosterone were higher in control rams than in immunized animals. The capacity of the antisera to bind 125I-labeled inhibin alpha-subunit increased significantly in each immunized animal within 30 days of treatment, even though neutralizing antibodies were detected with a rat pituitary cell culture bioassay in only one of the four immunized rams. Epididymal sperm reserves tended to be greater in immunized than in control animals. These results show that inhibin controls the release of FSH during the breeding season, thereby regulating spermatogenic activity; it may also exert its effect on testicular function by a local effect on Leydig cells, as evidenced by changes in serum testosterone profiles and increased serum LH levels in rams immunized against the inhibin alpha-subunit. 相似文献
109.
Steroid binding proteins bind steroid hormones with high affinity and their function is to carry those hormones in the extracellular compartment. Since their discovery more than fifty years ago, many reports concerning their physicochemical structures and functions have contributed to the better understanding of those proteins. Recent advances in recombinant DNA technology have led to the availability of molecular probes for these proteins, and new approaches have been used to analyse their gene structures as well as the regulation of their synthesis. In the present report, we will review the new findings of the last five years which include the cloning and sequencing of the cDNAs and genes for corticosteroid binding globulin, testosterone estradiol binding globulin and androgen binding protein, as well as the tissue distribution and regulation of their mRNAs in normal tissues and cancer cell lines. 相似文献
110.
An assay for quantifying the high affinity progesterone binding protein in guinea pig uterine cytosol was developed using Florisil to separate bound and free steroid. The activity of the progesterone binding protein increased between 4–12 hours following estrogen administration and by 4 days of treatment was 10-fold higher than castrate controls. When estrogen administration was discontinued the progesterone binding activity declined slowly with a half-life of 3 days. By contrast, progesterone treatment caused a rapid decline of binding activity within 3 hours. These studies suggest that the antagonistic actions of estrogen and progesterone determine the quantity of available progesterone binding sites in guinea pig uterine cytosol. 相似文献