To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability. 相似文献
Increasing evidence indicates that the gastrin-releasing peptide receptor (GRPR) is implicated in regulating synaptic plasticity
and memory formation in the hippocampus and other brain areas. However, the molecular mechanisms underlying the memory-impairing
effects of GRPR antagonism have remained unclear. Here we report that basic fibroblast growth factor (bFGF/FGF-2) rescues
the memory impairment induced by GRPR antagonism in the rat dorsal hippocampus. The GRPR antagonist [D-Tpi6, Leu13 psi(CH2NH)-Leu14] bombesin (6–14) (RC-3095) at 1.0 μg impaired, whereas bFGF at 0.25 μg enhanced, 24 h retention of inhibitory avoidance (IA)
when infused immediately after training into the CA1 hippocampal area in male rats. Coinfusion with an otherwise ineffective
dose of bFGF blocked the memory-impairing effect of RC-3095. These findings suggest that the memory-impairing effects of GRPR
antagonists might be partially mediated by an inhibition in the function and/or expression of neuronal bFGF or diminished
activation of intracellular protein kinase pathways associated with bFGF signaling. 相似文献
Platinated oligonucleotides are promising tools for the control of gene expression, since they may target and cross-link nucleic
acid chains. Here we describe a method for the preparation of platinated oligonucleotides that has proved able to selectively
cross-link complementary sequences, making use of 5-methylcytidine analogs with thioether or imidazole groups attached to
the 4-position. These nucleoside analogs were derivatized as phosphoramidites and introduced in oligonucleotide chains using
standard phosphite triester chemistry. Different oligonucleotide sequences containing either one or two analogs appending
from the 5′-end were synthesized and used in preliminary platination studies. The reaction of transplatin with oligonucleotides
containing the thioether-modified nucleobase was fast, but generally afforded unstable adducts and complex reaction mixtures.
The imidazole-containing oligonucleotides reacted with transplatin much more slowly, in particular at slightly basic pH, and
it was found that the imidazole-modified cytosine was less reactive than the natural nucleobases. In contrast, transplatin
selectively reacted with the thioether and imidazole groups of oligonucleotides containing the two cytosine analogs in neighboring
positions, even in the presence of the four nucleobases and particularly three guanines, affording platinated oligonucleotides
suitable for cross-linking.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
Faba beans are inexpensive, nutrient-dense sources of plant protein, but anti-nutritional factors such as condensed tannins
reduce the biological value of their protein. Two recessive genes, zt-1 and zt-2, control the absence of tannins in faba bean seeds and also determine a white flower character on the plant. However, crosses
between them produce coloured F1 plants with tannins that contaminate the crop. Therefore, it is important to identify the gene present in all tannin-free
cultivars and gene bank accessions to enable breeders to choose appropriate genitors for their crosses. The aim of this study
was the identification of markers linked to zt-1, one of the genes governing free tannin content in faba bean. A segregating F2 population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-1 line was developed and characterized phenotypically. Bulked Segregant Analysis (BSA) was used to identify Random Amplified
Polymorphic DNA (RAPD) markers linked to the zt-1 gene. Four RAPD loci (OPC5551, OPG15600, OPG111171 and OPAF20776) showed polymorphism between the contrasting bulks. The markers were sequenced to develop specific Sequence Characterised
Amplified Regions (SCARs). Amplification of SCC5551 produced a single product which was only observed in the white flowered and zero tannin content genotypes, whereas SCAR
SCG111171only produced a band in F2 plants with coloured flower and high tannin content. SCARs SCC5551 and SCG111171 were tested for their applicability for routine screening in 37 faba bean genotypes differing in flower colour and tannin
content. SCC5551, allowed the prediction of the zt-1 genotypes with a 95% of accuracy, underscoring the potential of this SCAR marker as a cost-effective tool for MAS in large
faba bean breeding populations. 相似文献
The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU?/? neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation.
Rodent ovariectomy is an experimental method to eliminate the main source of sexual
steroids. This work explored for the first time the ovariectomy temporal changes induced
in the hemostatic coagulation markers: prothrombin time (PT), activated partial
thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB) along
with uterine weight on adult female CD1 mice and Wistar rats. Uterine weight (Uw) was
assessed before ovariectomy (control), and 1, 3, 5, 7, 9, 16, and 21 days after surgery.
PT, aPTT, TT and FIB were estimated the same days, using reported standard techniques.
Ovariectomy decreased Uw, since day 1; and from day 10 to 21 reached the lowest values for
both species. After day 1, mice hemostatic parameters changed (PT +10%,
P<0.05; aPTT +53%, P<0.05; TT −24%,
P<0.05; FIB +67%, P<0.05). Rats showed
significant changes only in TT and FIB (TT −13%, P<0.001; FIB +65%,
P<0.001). Neither mice PT, aPTT and TT, recovered control values
after 21 days. In the rats from day 5 to 16 aPTT diminished (18–23%,
P<0.05) recovering to control values on day 21, TT after 9 days and PT
on day 16. In both species, FIB returned to its control values after 9 days. Ovariectomy
differentially altered the PT hemostatic parameter of mice and rats indicating a
non-equivalence among both species behaviour for experimental studies of blood
coagulation. 相似文献
The functional role of burst firing (i.e. the firing of packets of action potentials followed by quiescence) in sensory processing is still under debate. Should bursts be considered as unitary events that signal the presence of a particular feature in the sensory environment or is information about stimulus attributes contained within their temporal structure? We compared the coding of stimulus attributes by bursts in vivo and in vitro of electrosensory pyramidal neurons in weakly electric fish by computing correlations between burst and stimulus attributes. Our results show that, while these correlations were strong in magnitude and significant in vitro, they were actually much weaker in magnitude if at all significant in vivo. We used a mathematical model of pyramidal neuron activity in vivo and showed that such a model could reproduce the correlations seen in vitro, thereby suggesting that differences in burst coding were not due to differences in bursting seen in vivo and in vitro. We next tested whether variability in the baseline (i.e. without stimulation) activity of ELL pyramidal neurons could account for these differences. To do so, we injected noise into our model whose intensity was calibrated to mimic baseline activity variability as quantified by the coefficient of variation. We found that this noise caused significant decreases in the magnitude of correlations between burst and stimulus attributes and could account for differences between in vitro and in vivo conditions. We then tested this prediction experimentally by directly injecting noise in vitro through the recording electrode. Our results show that this caused a lowering in magnitude of the correlations between burst and stimulus attributes in vitro and gave rise to values that were quantitatively similar to those seen under in vivo conditions. While it is expected that noise in the form of baseline activity variability will lower correlations between burst and stimulus attributes, our results show that such variability can account for differences seen in vivo. Thus, the high variability seen under in vivo conditions has profound consequences on the coding of information by bursts in ELL pyramidal neurons. In particular, our results support the viewpoint that bursts serve as a detector of particular stimulus features but do not carry detailed information about such features in their structure. 相似文献
Engineering proteins for selective tissue targeting can improve therapeutic efficacy and reduce undesired side effects. The relatively high dose of recombinant human acid α-glucosidase (rhGAA) required for enzyme replacement therapy of Pompe disease may be attributed to less than optimal muscle uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR). To improve muscle targeting, Zhu et al. (1) conjugated periodate oxidized rhGAA with bis mannose 6-phosphate bearing synthetic glycans and achieved 5-fold greater potency in a murine Pompe efficacy model. In the current study, we systematically evaluated multiple strategies for conjugation based on a structural homology model of GAA. Glycan derivatives containing succinimide, hydrazide, and aminooxy linkers targeting free cysteine, lysines, and N-linked glycosylation sites on rhGAA were prepared and evaluated in vitro and in vivo. A novel conjugation method using enzymatic oxidation was developed to eliminate side oxidation of methionine. Conjugates derived from periodate oxidized rhGAA still displayed the greatest potency in the murine Pompe model. The efficiency of conjugation and its effect on catalytic activity were consistent with predictions based on the structural model and supported its use in guiding selection of appropriate chemistries. 相似文献
In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week. 相似文献