Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Instant success for Instant Pharmacology

Instant Pharmacology (1999). Saeb-Parsy K, Assomull RG, Kahn FZ, Saeb-Parsy K, and Kelly E. John Wiley and Sons 349 pp. £19.99 pbk; ISBN 0471976393 © 1999 John Wiley & Sons, Inc.     

人眼微光下夜近视机制的研究

本文利用激光散斑屈光测试系统详细探讨了夜近视(night myopia)的产生机制。夜近视在环境亮度降低到产生暗视觉时出现,并随亮度继续降低而增大,平均值为1.35D。实验证明眼睛球差和色差不是造成夜近视的主要因素。夜近视数值在暗适应过程中呈增大的趋势。在暗视觉时,由于视野中缺少适宜的刺激,眼睛处于暗焦(dark focus)休止状态,这是造成夜近视的主要原因。     

Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.     

Measurement of dose rate at the interface of cell culture medium and glass dishes by means of ESR dosimetry using thin films of alanine.

Previous studies on human cervical cancer cells (NHIK 3025) have indicated that the cells, when X-irradiated in suspension, appeared to be more radiosensitive than when they were irradiated attached to glass dishes. However, this result depends on dosimetry, which is difficult in the situation where cells are attached to glass dishes due to backscattering electrons at the glass-liquid interface. Recently developed dosimetry that is based on detection of radiation-induced stable radicals in alanine and uses ESR spectroscopy offers a possibility for more relevant dosimetry at the glass-liquid interface than the previous estimates of doses based on Fricke dosimetry. Thin alanine films (>/=10 microm) were used to measure dose at the interface by irradiating the films while they were placed tightly against the bottom of dishes and covered with 1 mm of wax simulating the medium above cells. Fricke dosimetry was also performed, with different depths of Fricke solution in the dishes, to elucidate the contribution to the dose delivered by backscattering electrons at the glass-liquid interface. A dose rate of 1.9 Gy/min was measured with a thin layer (0.2-0.3 mm) of Fricke solution in petri dishes made of glass. However, this estimate appears to be too high, due to a contribution to dose by short-ranged electrons generated when the X rays passed through a steel lid 4.5 cm above the dishes. Dosimetry using alanine films resulted in dose rates of 1.15 and 0.87 Gy/min at the interfaces of glass-liquid and plastic- liquid, respectively. Hence there is a significant contribution to dose from backscattering electrons on dishes made of glass. The reason for our previous observation of a difference in radiosensitivity between cells irradiated in suspension and cells irradiated attached to glass appears to be a lack of accurate dosimetry at the glass-liquid interface.     

瓜类枯萎病研究进展(综述)

瓜类枯萎病是由尖孢镰刀菌Fusarium oxysporum侵染引起,在全球大部分瓜类产区均有发生。该病已成为影响我国瓜类品质和产量的重要病害。本文从瓜类作物枯萎病的防治方法、致病机理和分子生物学研究等方面综述其研究现状,并对今后的研究方向进行展望。     

beta1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T) expression in normal rat tissues and different cell lines: evidence for complex mechanisms of regulation

The distribution of the Golgi enzyme beta1, 6-N- acetylglucosaminyltransferase (core 2 GlcNAc-T for short) has been investigated in several tissue and cell systems by combining the potentials of a polyclonal antibody and a novel, sensitive fluorescent enzyme assay. In normal rat tissues, levels of the protein were found to vary and as a general trend did not correlate with enzyme activities. Additionally, we observed tissue-specific core 2 GlcNAc-T forms of various size: 75 kDa (liver), 70 kDa (spleen), 60 kDA (heart), and 50 kDa (heart and lung). These forms might arise from differential protein modifications; alternatively, the smaller form may be a product of proteolytic cleavage, given the presence of a catalytically inactive 50 kDa species in rat serum. Chinese hamster ovary (CHO), MDAY-D2, PSA- 5E, and PYS-2 cell lines consistently displayed a 70 kDa enzyme. When induced to retrodifferentiate in the presence of butyrate + cholera toxin, CHO cells exhibited a 21-fold increase in enzyme activity, while protein levels remained constant. A similar trend was observed in the embryonal endoderm cell lines PSA-5E and PYS-2, where an approximately 100-fold difference in core 2 GlcNAc-T activity was found notwithstanding unchanged amounts of the protein and identical mRNA levels, as evidenced by RT-PCR. In contrast, levels of core 2 GlcNAc-T activity in MDAY-D2 cells correlated well with protein expression. Taken together, these observations demonstrate that core 2 GlcNAc-T expression may be subjected to multiple mechanisms of regulation and suggest that in at least some instances (i.e., PSA-5E and PYS-2 cells) expression may be regulated exclusively via posttranslational mechanism(s) of control.      

人胎儿脑的一个cDNA编码序列及其蛋白质预测

用 m RNA差异显示 PCR技术 ,从人 1 8周、2 2周胎儿脑和肝肾组织的 m RNA逆转录产物得到一些特异性显示的片段 .其中一个随机片段 GC1 0 2作为探针 ,从本实验室构建的 1 8周胎儿脑c DNA基因文库进行杂交筛选 ,得到一个阳性克隆λ gt1 0 /GC1 0 2 .该克隆内插入的 c DNA片段长2 .9kb,经过 DNA测序 ,显示具有一个开放阅读框架 ,编码 1 37个氨基酸的肽链 .用蛋白质结构的建模软件预测了该肽链的立体结构初步模型 .     

基于PBL模式的生物化学教学探索

目的:评价PBL教学模式在生物化学中应用的教学效果,对生物化学理论教学的方式进行探讨。方法:用PBL模式进行教学,采用理论考试的形式对教学效果进行评价。结果:PBL教学组的学生在学习态度和积极性等方面好于传统教学组;在考试成绩方面PBL教学组病例分析考试成绩好于传统教学组,而基础知识考试成绩没有差别。结论:PBL模式能提高学生的分析问题和解决问题的综合应用能力,而基础知识的掌握程度与传统教学相比没有区别。