Combining spin echoes with gradient echoes in the context of the global coherent free precession pulse sequence.

To extend the signal longevity of magnetically excited spins in flowing fluids while in a state of global coherent free precession (GCFP), a refocusing radiofrequency (RF) pulse and bipolar gradient waveforms were combined with the GCFP sequence. The data demonstrate that RF refocusing in the presence of flowing blood is possible, but the improvement in signal amplitude depends on the static magnetic field homogeneity along the direction of motion and the displacement of the spins between the excitation and the RF refocusing pulse, as well as displacement during subsequent RF refocusing pulses. The least amount of phase dispersion and thus the longest lasting signal is obtained with the shortest echo spacing where only one line of data is recorded between two RF refocusing pulses. This approach was successfully used in a phantom and in vivo to image fast and slow blood flow. Depending on the experimental conditions, signal persistence is improved significantly compared to playing the same sequence without RF refocusing, but the improvement is limited by the product of blood flow velocity and the time between RF refocusing pulses.     

应用激光显微分离技术在骨肉瘤组织原位筛选噬菌体肽库

目的将激光显微分离(laser capture microdissection, LCM) 技术应用于噬菌体表面呈现肽库的筛选过程,建立一种可以直接在天然组织中筛选肽库的方法。方法将新鲜人骨肉瘤组织块在噬菌体肽库溶液振荡孵育后制成组织冰冻切片,免疫组化染色检测噬菌体在组织中的浸润扩散。改进常规LCM切片处理方法,以冻干法替代酒精/二甲苯法脱水,以期在LCM操作过程中提高切片上噬菌体的存活率。LCM法分离摄取骨肉瘤切片上的肿瘤靶细胞,转染回收这些细胞上特异结合的噬菌体。滴定法检测所筛选的噬菌体对特异性细胞的亲和力。结果利用LCM技术,可以由肿瘤冰冻切片上收集到足够的与特异性噬菌体短肽,应用于噬菌体表面呈现肽库筛选。经过3轮筛选后所获得的噬菌体与人骨肉瘤组织的特异性亲和力提高16倍。结论本研究首次将LCM技术应用于噬菌体表面呈现肽库的筛选,可以使我们直接在新鲜人肿瘤组织中筛选与特定细胞群甚至单个细胞亲合的短肽;同时又避免了天然组织中其他杂质细胞的污染,为研制细胞特异性导向载体提供了一个新的途径。     

诱生型一氧化氮合酶特异性抑制肽的制备

目的：通过噬菌体展示技术筛选iNOS特异性抑制肽。方法：将iNOSFAD结合区及其附近序列的基因片段装入pET-28A( )，在大肠杆菌BL21中表达，His.bind^TM亲和层析柱纯化目的蛋白，使用纯化蛋白筛选Ph.D.-12^TM噬菌体库，筛选iNOS活性抑制作用较高的噬菌体克隆，测序并合成其中具有一致序列的短肽。结果：得到具有较高表达量的目的蛋白，经His.Bind^TM柱亲和层析纯化后纯度大于95％，以纯化蛋白筛选Ph.D.-12^TM噬菌体库，经4轮筛选获得10株iNOS活性抑制作用较高的噬菌体克隆，测序发现其中5株序列完全相同，合成该12肽，初步研究表明其对iNOS表现为高浓度抑制，低浓度兴奋的作用，而对nNOS及eNOS则没有影响。结论：以iNOSFAD片段蛋白为靶蛋白筛选得到的克隆对iNOS活性具有特异性影响，可根据这些特征设计合成小分子前导药物，创造新的活性药物。     

RASER: a new ultrafast magnetic resonance imaging method.

A new MRI method is described to acquire a T(2)-weighted image from a single slice in a single shot. The technique is based on rapid acquisition by sequential excitation and refocusing (RASER). RASER avoids relaxation-related blurring because the magnetization is sequentially refocused in a manner that effectively creates a series of spin echoes with a constant echo time. RASER uses the quadratic phase produced by a frequency-swept chirp pulse to time-encode one dimension of the image. In another implementation the pulse can be used to excite multiple slices with phase-encoding and frequency-encoding in the other two dimensions. The RASER imaging sequence is presented along with single-shot and multislice images, and is compared to conventional spin-echo and echo-planar imaging sequences. A theoretical and empirical analysis of the spatial resolution is presented, and factors in choosing the spatial resolution for different applications are discussed. RASER produces high-quality single-shot images that are expected to be advantageous for a wide range of applications.     

Effect of amyloid peptides on the increase in TrkA receptor expression induced by nicotine in vitro and in vivo

The ability of nicotine to induce a cytoprotective or neuroprotective action occurs through several down-stream mechanisms. One possibility is that the drug increases the expression of tyrosine kinase A (TrkA) nerve growth factor (NGF) receptors. Certain β-amyloid peptides (e.g., Aβ1–42) have been shown to bind with high affinity to α7 nicotinic receptors and thus interfere with a potentially neurotrophic influence. Treatment of differentiated PC-12 cells with nicotine produced a concentration-dependent increase in cell-surface TrkA receptors that occurred concomitantly with cytoprotection. The effect of nicotine was blocked by either of the α7 receptor antagonists α-bungarotoxin (α-BTX) or methyllycaconatine. The cytoprotective action of nicotine also was inhibited by pretreatment with 10–100 nM Aβ1–42. Nicotine also was administered (four injections of 30 μg, spaced evenly over 24 h) to rats by direct injection into a lateral cerebral ventricle. Brain TrkA expression was increased significantly in hippocampus and entorhinal cortex (up to 32% above control), with no changes found in cerebral cortex or hypothalamus. The nicotine-induced increases in TrKA expression in hippocampus and entorhinal cortex were significantly inhibited by 10 μg α-BTX or by 10 nmol Aβ1–42. Therefore, physiologically relevant concentrations of Aβ1–42 can prevent nicotine-induced TrkA receptor expression in brain regions containing cholinergic neurons susceptible to the neurotoxicity associated with Alzheimer’s disease.     

对数期继代对南方红豆杉细胞培养动力学的影响

目的 解决南方红豆杉细胞生长缓慢及代谢水平低下的问题。方法 对对数期(20d)和静止期(30d)的红豆杉细胞分别进行继代，在整个生长周期中测定了培养基中的碳源、氮源、磷酸盐的变化并分析了红豆杉细胞生长及紫杉醇的合成情况。结果 对数期继代细胞吸收碳源和硝态氮早于静止期继代的细胞，且前者的比生长速度是后者的1．5倍，紫杉醇含量提高了近4倍。结论 对数期继代有利于生物量的积累及紫杉醇的合成。     

Ca2+ mobilization in physiologically stimulated single T cells gradually increases with peptide concentration (analog signaling)

We have investigated Ca²⁺ mobilization in single T cells stimulated with their physiological ligand, i.e. antigenic peptide bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). Fibroblasts expressing I-E^d class II molecules were pulsed with a peptide derived from the λ2³¹⁵ immunoglobulin light chain. Onto such antigen-pulsed fibroblasts were sedimented cloned Th1 cells loaded with Fura-2. Changes in cytosolic Ca²⁺ concentration in single T cells were continually monitored by use of an imaging system based on fluorometry. Ca²⁺ mobilization was both peptide-specific and MHC-restricted. Within seconds of the initial APC-T cell contact, a Ca²⁺ spike could be observed. The Ca²⁺ response gradually declined over a 25-min period, during which oscillations were noted. Various parameters characterizing the magnitude of the Ca²⁺ response (latency, increase rate, max and mean Ca²⁺ increase, frequency and period of oscillations) all correlated with the amount of peptide used for pulsing the fibroblasts. Thus, Ca²⁺ mobilization in single T cells appears not to be an all or none phenomenon. Rather, activation is incremental (analog signaling), the degree of Ca²⁺ mobilization probably being related to the number of stimulatory peptide-MHC complexes on the surface of the APC. The extent of calcium mobilization and lymphokine production (interleukin (IL)-2, IL-3, interferon-γ) correlated, at least at the population level.     

Seasonal covariation of the circadian phases of rectal temperature and slow wave sleep onset

There is a scarcity of well-controlled studies of the seasonal variation in circadian rhythmicity. In the present study, the circadian phase of rectal temperature and the onset of slow wave sleep were studied in a series of twelve 24-h experiments, one each month of the year, for six healthy subjects under controlled conditions in a climatic chamber. In winter, as compared with summer, the average circadian rhythm of rectal temperature was phase delayed by 45 min, and the average onset of slow wave sleep was phase delayed by 40 min. The temporal relationship between the circadian phase of rectal temperature and the timing of slow wave sleep was maintained throughout the year. Habitual rising and retiring times covaried as well. Furthermore, the circadian rhythm of rectal temperature followed the timing of the photoperiod across the year, but had a much smaller range of seasonal variation. Apparently, the seasonal variation in the photoperiodic zeitgeber is largely compensated for by the stabilizing influence of secondary zeitgebers. However, in healthy subjects some effect of photoperiodic variation can still be observed.     

Effects of bombesin and gastrin-releasing peptide on memory processing

We have previously shown that feeding mice immediately following training enhances memory retention and that one of the gastrointestinal hormones released during a meal, cholecystokinin, also enhances retention after peripheral administration. In the studies reported here we demonstrate that another gastrointestinal peptide, gastrin-releasing peptide (GRP), enhances retention after peripheral administration, as does its amphibian counterpart, bombesin. GRP_14–27 had the same effect as the intact peptide, while GRP_1–16 was ineffective at enhancing retention. The dose-response curves showed a characteristic inverted U-shape with high doses of both GRP and bombesin being amnestic. The effect of both peptides was time-dependent and both reversed amnesia induced by the anticholinergic, scopolamine. I.c.v. administration of the peptides required higher doses to produce an effect on memory retention. suggesting that the effect was mediated predominantly through a peripheral mechanism. Doses of the peptides that enhanced memory retention after peripheral administration failed to increase serum glucose, suggesting that glucose modulation was not the mechanism by which GRP and bombesin modulate memory processing. Vagotomy inhibited the memory-enhancing effects of both GRP and bombesin, suggesting that these peptides produced their effect by stimulating ascending vagal pathways. These studies, together with our previous study with cholecystokinin, suggest the existence of a gastrointestinal hormonal system, which is activated by the passage of food through the intestine, that enhances memory retention.     

Neutrophilic defensins penetrate the blood-brain barrier

Defensins are small, cationic, cyclic peptides that are abundantly stored in granules of neutrophils. Defensins non-specifically interact with membranes by forming weakly ion-selective pores. Here we demonstrate immunolocalization of defensin-secreting cells in human brain. Defensins, secreted by activated granulocytes, apparently are not prevented by the blood-brain barrier (BBB) from diffusing across cerebral endothelium to penetrate the neuropil for a considerable distance from the granulocyte. This is in contrast to other neutrophil proteins like the granuleassociated enzyme elastase or the cytosolic protein MRP-14, which are strictly localized to the cytoplasm or granules of neutrophils. Thus, defensins, known chemokinetic and chemotactic molecules, display a unique distribution at BBB sites. © 1995 Wiley-Liss, Inc.