Spontaneous and MNNG‐induced reversion of an EGFP construct in HeLa cells: An assay for observing mutations in living cells by fluorescent microscopy

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2‐654 intron, was studied without treatment and after treatment with a single standard dose of 15 μM of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non‐mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (–62, –100, and –162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G₆₅₂, G₆₅₃, A₆₅₅, G₅₇₉), and a pyrimidine (T₆₅₄) between nucleotide positions 579 and 837. Splice‐site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay. Hum Mutat 18:526–534, 2001. © 2001 Wiley‐Liss, Inc.     

激光扫描共聚焦显微镜在肿瘤研究中的应用

激光扫描共聚焦显微镜是近年来出现的一种新的分子细胞生物学分析仪器,已经广泛应用于生物医学研究的许多领域.简介其基本功能和特点.并从肿瘤组织细胞蛋白的定位和定量、肿瘤细胞亚细胞结构观察、肿瘤受体研究、肿瘤药物分布和肿瘤多耐药机制等角度.综述了近年来激光扫描共聚焦显微镜在肿瘤研究中应用的进展.     

The effects of chronic papillary necrosis on acid excretion

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2±2.1 vs. 31.5±3.6% filtering, control vs. BEA, respectively,P<0.001) and significantly reduced in the superficial nephrons (80.6±3.6 vs. 62.2±6.1% filtering, control vs. BEA, respectively,P<0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH₄Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na₂SO₄ infusion and phosphate infusion was the same in both groups of animals. The urineblood (U-B)pCO₂, an index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.     

共聚焦激光扫描显微镜(MRC600)活体观测川芎嗪和去甲基肾上腺素对休克状态下家兔大脑皮质内微循环的影响

目的 :共聚焦激光扫描显微镜活体观测川芎嗪和去甲基肾上腺素对休克状态下家兔大脑皮质内微循环的影响。方法 :在开放颅窗的家兔模型上 ,荧光素标记血浆 ,罗丹明 6G标记WBC ,用共聚焦激光扫描显微镜活体观测川芎嗪和去甲基肾上腺素对休克状态下家兔大脑皮质内微循环的影响 ,并经图像分析系统测量数据 ,用SAS软件包进行统计学分析。结果 :①川芎嗪抗休克效果优于去甲基肾上腺素 ;②去甲基肾上腺素在休克状态下对口径为 60 .15 μm的动脉血管处未引起明显的血管运动 ,而川芎嗪能引起血管运动 ,尤以大剂量川芎嗪引起强烈的血管运动 ;③川芎嗪和去甲基肾上腺素增加或保持血液缘流厚度不变 ,可能是两者抗休克机制发挥作用的途径之一 ;④川芎嗪和去甲基肾上腺素引起血管运动 ,尤以中小血管处明显。结论 :川芎嗪抗休克效果优于去甲基肾上腺素。川芎嗪和去甲基肾上腺素增加或保持血液缘流厚度不变 ,可能是两者抗休克机制发挥作用的途径之一     

Generators of pathologically enhanced excitation as determinant structures in the spinal cord

With the aid of tetanus toxin, which disturbs various types of inhibition, generators of excitation were created in the left and right anterior horns of the lumbar spinal cord in rats. The regimes of activity of the generators differed: the left-sided generator, formed during the longer action of the toxin, in response to activation by trigger stimulation first produced tonic, and then intermittent activity, or individual spontaneous discharges, whereas the righ-sided generator produced only tonic activity. If one generator was blocked by glycine, the other continued to operate as before. Activation of one generator led to concomitant depression of the effects of the other. During separate activation of each generator, all the spinal and supraspinal motoneuron pools synchronously reproduced the character of activity of the generator functioning at that particular moment. The generator thus played the role of a determinant structure, determining the behavior of the system. The results are examined from the standpoint of the general concept of the role of determinant structures in the activity of the nervous system and the theory of generator mechanisms of neuropathological syndromes characterized by hyperactivity of systems.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 515–519, May, 1977.     

硫化氢对体外大鼠肝星状细胞内Ca2+浓度变化及细胞增殖的影响

目的 研究硫化氢(H₂ S)对大鼠肝星状细胞-T6(HSC-T6) Ca²⁺浓度、细胞增殖的影响及其机制。 方法 活化HSC-T6用含10%小牛血清DMEM培养液制备为1×10⁵个肝星状细胞(HSC)悬液。钙离子荧光探针Fluo-3/AM负载细胞后，在不同刺激条件下，利用激光扫描共焦显微镜动态扫描HSC-T6细胞内Ca²⁺荧光强度（FI）变化，FI表示细胞内Ca²⁺浓度。四唑盐比色法，观察不同浓度H₂S供体——NaSH对HSC-T6细胞增殖的影响。 结果 低浓度H₂S（100μmol/L）明显降低HSC-T6细胞内Ca²⁺浓度（P<0.05)，而细胞增殖增加（增殖率为116%）；K_ATP通道阻断剂——格列本脲可阻断H2S的作用。高浓度H₂S（1mmol/L）刺激HSC-T6细胞内Ca₂₊浓度增加，但细胞增殖无明显变化(P＞0.05)。 结论 低浓度H₂S通过激活HSC-T6细胞K_ATP通道降低细胞内Ca²⁺浓度，可能通过调节细胞氧化应激促进细胞增殖；高浓度H2S刺激HSC-T6细胞内Ca₂₊浓度增加。提示H₂S在肝硬化门脉高压症的发生机制中具有双重作用。     

Segmental heterogeneity of swelling-induced Cl transport in rat small intestine

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (I _sc) and potential difference (V ^ms) in the jejunum, whereas the ileum responded with an increase in I _sc and V ^ms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl⁻ and was blocked by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in I _sc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl⁻ secretion induced by hypotonicity was blocked by the K⁺ channel blocker quinine and was reversed into a decrease in I _sc when serosal Ca²⁺ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl⁻ channels and in the ileum by a Ca²⁺-mediated opening of K⁺ channels which enhances apical Cl⁻ efflux. Received: 27 June 1995/Received after revision: 8 December 1995/Accepted: 28 December 1995     

人椎间盘髓核细胞突起的形态学特征

目的 探讨成人腰椎间盘髓核细胞突起的形态学特征.方法 取8例成人腰椎间盘髓核组织标本(Thompson Ⅰ～Ⅱ),分别行冰冻切片和电镜切片,同时进行髓核细胞的分离和单层培养,利用光镜、激光扫描共焦显微镜和透射电镜,从组织、细胞和超微结构水平观察细胞突起的形态学特征.结果 所有的髓核细胞均具有明显的突起结构,相邻的细胞突起间可见缝隙连接.在体状态下均呈类圆形的髓核细胞进行离体培养时却呈现梭形和类圆形两种不同的形态,梭形细胞与类圆形细胞的比例约为2.3∶1.梭形细胞的突起顺着细胞体长轴发出,未见二级突起.类圆形细胞的突起从细胞体四周发出,突起呈树枝状,可见多级突起.结论 突起是椎间盘髓核细胞的形态学特征之一,对突起功能的深入研究将有助于加深对椎间盘退变病理机制的认识.     

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.