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排序方式: 共有122条查询结果,搜索用时 15 毫秒
81.
A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling. 总被引:2,自引:0,他引:2
Ultracryotomy of fixed specimens in combination with immunogold labeling is widely used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible. Normally, sections are cut and labeled within 1 day to prevent possible loss or redistribution of soluble antigens within the sections. An increasing demand for more sections and multiple labeling protocols prompted us to investigate the extent to which ultrathin cryosections can be stored. This would render the time spent behind an ultracryomicrotome more efficient and would allow immunogold labeling at a later stage. We investigated whether gelatin plates, 2.3 M sucrose, or 1.0% methyl cellulose/1.2 M sucrose can be used to store thawed frozen sections for a longer period of time. Ultrathin sections of mildly fixed tissue and cultured cells were stored for up to 6 months before immunogold labeling. The preservation of the ultrastructure of stored sections was excellent and was similar to that of immediately processed sections. Importantly, prolonged storage did not affect the labeling intensity. 相似文献
82.
Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA, from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System 总被引:1,自引:0,他引:1 下载免费PDF全文
Clara C. Posthuma Rechien Bader Roswitha Engelmann Pieter W. Postma Wolfgang Hengstenberg Peter H. Pouwels 《Applied microbiology》2002,68(2):831-837
Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA. 相似文献
83.
de Bruin JP Dorland M Spek ER Posthuma G van Haaften M Looman CW te Velde ER 《Biology of reproduction》2004,70(2):419-424
Age-related decline of fertility in women is the result of the decline in both quantity and quality of the resting ovarian follicle pool. The aim of the present study was to determine whether the decline of follicle quality with age is reflected by ultrastructural changes in the resting follicle pool. Ovarian biopsy specimens were obtained by laparoscopy from seven healthy women aged 25-32 yr (young group) and from 11 healthy women aged 38-45 yr (advanced-age group). A total of 182 resting follicles from the young group were compared with 81 resting follicles from the advanced-age group for signs of age-related changes by transmission-electron microscopy. The ooplasmic fraction of vacuoles was increased (P = 0.02), and the fraction of mitochondria decreased (P = 0.005), in the advanced-age group. Also, the density of the mitochondrial matrix (P < 0.001) and the frequency of dilated smooth endoplasmic reticulum (SER; P = 0.001) and Golgi complex (P = 0.02) were increased with age. The frequencies of ruptured mitochondrial membranes (P = 0.001) and dilated SER (P = 0.003) were increased with age in the granulosa cells. Overall follicle-quality scores, which should reflect atretic changes, were not different for the young and advanced-age groups. In conclusion, in resting follicles, the morphological changes with age are different from the changes seen in quality decline by atresia. The morphological changes with age specifically involved the mitochondria, the SER, and the Golgi complex, and they may be the cause of atresia on initiation of follicular growth because of the substantial increase in metabolic requirements. 相似文献
84.
de Bruin JP Dorland M Spek ER Posthuma G van Haaften M Looman CW te Velde ER 《Biology of reproduction》2002,66(4):1151-1160
In humans, follicle quantity and quality decline with age by atresia. In the present study we aimed to describe the quality of the follicle pool through an ultrastructural investigation of resting follicles in young healthy women. From ovarian biopsies of 7 women aged 25-32 yr, 182 small follicles were morphometrically assessed for various signs of atresia. Morphometric variables were analyzed by principal components analysis (PCA) to demonstrate correlations between variables and to construct an objective follicle score. One third of small follicles consisted of primordial follicles. Nucleus:cell ratios remained constant for oocytes and granulosa cells from primordial to primary follicles, suggesting that follicles up to primary stages belong to the resting pool. The distribution of follicle quality scores as derived from PCA showed that most follicles were of good quality and with little signs of atresia. Atresia in resting follicles appears to be a necrotic process, starting in the ooplasma. Early atresia was characterized by increasing numbers of multivesicular bodies and lipid droplets, dilation of smooth endoplasmic reticulum and Golgi, and irregular mitochondria with changed matrix density. In progressive atresia mitochondrial membranes ruptured, oocyte nuclear membranes were indented or ruptured, and the ooplasma showed extensive vacuolarization. The early involvement of mitochondria in this process suggests that damage is induced by oxygen radicals. PCA follicle quality scores can be reliably approximated using a reduced number of seven morphometric variables, which were selected by stepwise forward analysis. The algorithm to calculate these follicle scores is presented. 相似文献
85.
A genomewide scan for intelligence identifies quantitative trait loci on 2q and 6p 总被引:10,自引:0,他引:10 下载免费PDF全文
Posthuma D Luciano M Geus EJ Wright MJ Slagboom PE Montgomery GW Boomsma DI Martin NG 《American journal of human genetics》2005,77(2):318-326
Between 40% and 80% of the variation in human intelligence (IQ) is attributable to genetic factors. Except for many rare mutations resulting in severe cognitive dysfunction, attempts to identify these factors have not been successful. We report a genomewide linkage scan involving 634 sibling pairs designed to identify chromosomal regions that explain variation in IQ. Model-free multipoint linkage analysis revealed evidence of a significant quantitative-trait locus for performance IQ at 2q24.1-31.1 (LOD score 4.42), which overlaps the 2q21-33 region that has repeatedly shown linkage to autism. A second region revealed suggestive linkage for both full-scale and verbal IQs on 6p25.3-22.3 (LOD score 3.20 for full-scale IQ and 2.33 for verbal IQ), overlapping marginally with the 6p22.3-21.31 region implicated in reading disability and dyslexia. 相似文献
86.
87.
Zn(2+) modulation of neuronal transient K(+) current: fast and selective binding to the deactivated channels 总被引:1,自引:0,他引:1 下载免费PDF全文
Modulation of voltage-dependent transient K(+) currents (A type K(+) or K(A) current) by Zn(2+) was studied in rat hippocampal neurons by the whole-cell patch-clamp technique. It is found that Zn(2+) selectively binds to the resting (deactivated or closed) K(A) channels with a dissociation constant (K(d)) of approximately 3 &mgr;M, whereas the affinity between Zn(2+) and the inactivated K(A) channels is 1000-fold lower. Zn(2+) therefore produces a concentration-dependent shift of the K(A) channel inactivation curve and enhances the K(A) current elicited from relatively positive holding potentials. It is also found that the kinetics of Zn(2+) action are fast enough to compete with the transition rates between different gating states of the channel. The rapid and selective binding of Zn(2+) to the closed K(A) channels keeps the channel in the closed state and explains the ion's concentration-dependent slowing effect on the activation of K(A) current. This in turn accounts for the inhibitory effect of Zn(2+) on the K(A) current elicited from hyperpolarized holding potentials. Because the molecular mechanisms underlying these gating changes are kinetic interactions between the binding-unbinding of Zn(2+) and the intrinsic gating processes of the channel, the shift of the inactivation curve and slowing of K(A) channel activation are quantitatively correlated with ambient Zn(2+) over a wide concentration range without "saturation"; i.e., The effects are already manifest in micromolar Zn(2+), yet are not saturated even in millimolar Zn(2+). Because the physiological concentration of Zn(2+) could vary over a similarly wide range according to neural activities, Zn(2+) may be a faithful physiological "fine tuner," controlling and controlled by neural activities through its effect on the K(A) current. 相似文献
88.
89.
Wim M.M. Schaaper Roger A.H. Adan Truus A. Posthuma Julia Oosterom Willem-Hendrik Gispen Rob H. Meloen 《Letters in Peptide Science》1998,5(2-3):205-208
Cyclic lactam analogs of -melanocyte stimulating hormone (-MSH) have been shown to be potent agonists in the frog skin bioassay [Al-Obeidi, F. et al., J. Med. Chem., 32 (1989) 2555], demonstrating melanocortin-1 (MC1) receptor activity. We synthesized cyclic -MSH(1-13) and -MSH(4-10) lactam analogs. The peptides were synthesized using Fmoc chemistry. We improved the cyclization procedure: side chains of Asp5 and Lys10 from the deprotected peptide were coupled in DMF to form a cyclic lactam, using an excess of PyBOP reagent and DIEA as a base. The cyclization reaction was completed within 1 h and was almost quantitative. We also synthesized an -MSH analog cyclized via a disulphide bridge. The peptides were tested for their selectivity for the rat MC4 receptor. Cyclization and substitutions at position 7 dramatically influenced the selectivity for the rMC4 receptor. 相似文献
90.
John S. Woods Mattia Damiani Peter Fantke Andrew D. Henderson John M. Johnston Jane Bare Serenella Sala Danielle Maia de Souza Stephan Pfister Leo Posthuma Ralph K. Rosenbaum Francesca Verones 《The International Journal of Life Cycle Assessment》2018,23(10):1995-2006