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41.
Papac  DI; Briggs  JB; Chin  ET; Jones  AJ 《Glycobiology》1998,8(5):445-454
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   
42.
Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.  相似文献   
43.
Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster   总被引:9,自引:0,他引:9  
A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.   相似文献   
44.

Key message

This study compares five models of GWAS, to show the added value of non-additive modeling of allelic effects to identify genomic regions controlling flowering time of sunflower hybrids.

Abstract

Genome-wide association studies are a powerful and widely used tool to decipher the genetic control of complex traits. One of the main challenges for hybrid crops, such as maize or sunflower, is to model the hybrid vigor in the linear mixed models, considering the relatedness between individuals. Here, we compared two additive and three non-additive association models for their ability to identify genomic regions associated with flowering time in sunflower hybrids. A panel of 452 sunflower hybrids, corresponding to incomplete crossing between 36 male lines and 36 female lines, was phenotyped in five environments and genotyped for 2,204,423 SNPs. Intra-locus effects were estimated in multi-locus models to detect genomic regions associated with flowering time using the different models. Thirteen quantitative trait loci were identified in total, two with both model categories and one with only non-additive models. A quantitative trait loci on LG09, detected by both the additive and non-additive models, is located near a GAI homolog and is presented in detail. Overall, this study shows the added value of non-additive modeling of allelic effects for identifying genomic regions that control traits of interest and that could participate in the heterosis observed in hybrids.
  相似文献   
45.
Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.  相似文献   
46.
Kinetics of veratridine action on Na channels of skeletal muscle   总被引:15,自引:8,他引:7       下载免费PDF全文
Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.  相似文献   
47.
Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.  相似文献   
48.
Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.  相似文献   
49.
Maternal parasitoses modulate fetal immune development, manifesting as altered cellular immunological activity in cord blood that may be linked to enhanced susceptibility to infections in early life. Plasmodium falciparum typifies such infections, with distinct placental infection-related changes in cord blood exemplified by expanded populations of parasite antigen-specific regulatory T cells. Here we addressed whether such early-onset cellular immunological alterations persist through infancy. Specifically, in order to assess the potential impacts of P. falciparum infections either during pregnancy or during infancy, we quantified lymphocyte subsets in cord blood and in infants'' peripheral blood during the first year of life. The principal age-related changes observed, independent of infection status, concerned decreases in the frequencies of CD4+, NKdim and NKT cells, whilst CD8+, Treg and Teff cells'' frequencies increased from birth to 12 months of age. P. falciparum infections present at delivery, but not those earlier in gestation, were associated with increased frequencies of Treg and CD8+ T cells but fewer CD4+ and NKT cells during infancy, thus accentuating the observed age-related patterns. Overall, P. falciparum infections arising during infancy were associated with a reversal of the trends associated with maternal infection i.e. with more CD4+ cells, with fewer Treg and CD8+ cells. We conclude that maternal P. falciparum infection at delivery has significant and, in some cases, year-long effects on the composition of infants'' peripheral blood lymphocyte populations. Those effects are superimposed on separate and independent age- as well as infant infection-related alterations that, respectively, either match or run counter to them.  相似文献   
50.

Background

Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC) in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6ε domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6ε domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates.

Methodology/Principal Findings

Local alignment analysis of the DBL6ε domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6ε domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI). Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6ε domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence.

Conclusions/Significance

A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types. Based on peptide based ELISA, variable blocks with 85% or greater sequence identity are expected to be recognized equally well by antibody and can be considered the same consensus type. Therefore, the analysis of the antibody response against the classified small number of sequences should be helpful to determine epitopes.  相似文献   
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