An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.     

核微管组织中心的研究

本文利用间接免疫荧光手段对间期核微管组织中心的性质进行了初步探讨。当分离核与6S微管蛋白保温后,用抗管蛋白抗体进行间接免疫荧光染色,在群体中只有极少数核上有微管生长。Triton X-100或溶血卵磷脂去膜核的实验证实能长微管的核是核膜缺失或破损核。用秋水仙素解聚核上长出的微管,可见核内荧光亮点数多于染色体数,说明核内微管组织中心数目多于着丝粒数,很可能包括着丝粒和一部分染色质颗粒。将秋水仙素和6S微管蛋白混合后与去膜核保温,结果6S微管蛋白与核内微管组织中心有亲和力。本文就这些结果进行了讨论。     

Platelet-activating factor antagonists increase vascular reactivity in perfused rat lungs

Platelet-activating factor (PAF) administered to the pulmonary circulation in low dose (nanogram) has vasodilatory properties. Therefore, we investigated whether endogenous PAF plays a role in the control of tone in the pulmonary circulation. The PAF receptor antagonists, SRI 63-441 (2.6 X 10(-4) M) and L659,989 (1 X 10(-5) M), were the major investigative tools. In isolated perfused rat lungs, both agents caused a persistent increase in base-line perfusion pressure (Ppa), potentiated angiotensin II (ANG II) vasoconstriction, and potentiated hypoxic vasoconstriction (HPV). This potentiation of ANG II and HPV was found to be independent of circulating blood elements. Vasodilation in the presence of PAF blockade was also impaired. The combination of cyclooxygenase inhibition and PAF receptor blockade had an additive effect on ANG II vasoconstriction but did not cause more potentiation of HPV than achieved with PAF antagonism alone. In vivo, SRI 63-441 (10 mg/kg) caused only a transient increase in base-line Ppa without altering ANG II and hypoxic vasoconstriction. These findings support a vasodilatory role for endogenous PAF in the pulmonary circulation.     

Effect of metabolic or respiratory acidosis on rabbit renal medullary proton-ATPase

Distal urinary acidification is thought to be mediated by an H+-ATPase sensitive to N-ethylmaleimide and dicyclohexyl-carbodiimide. We have studied the effect of chronic metabolic acidosis (NH4Cl for 3 days) or respiratory acidosis (inhalation of 10% CO2 for 2 days) on the H+-ATPase of plasma membranes prepared from the medulla. The enzymatic assay for the H+-ATPase was performed in the presence of ouabain and oligomycin and in the absence of Ca. H+-transport activity was assessed by the quenching of acridine orange in the presence of ATP. The 15-25% sucrose gradient fraction was enriched 40-fold in enzymatic activity over the homogenate, and 8-fold in enzymatic activity and 4-fold in H+-transport activity over the fluffy fraction (38,000 X g). Metabolic acidosis (pH less than 7.31) or chronic hypercapnia (PCO2 greater than 66 mmHg; 1 mmHg = 133.3 Pa) was induced for 2-3 days. Both groups showed the same enrichment factor in enzymatic and H+-transport assays as the control rabbits. Enzymatic and H+-transport activities, however, were not different between animals with respiratory acidosis and controls. Kinetic studies failed to disclose an increase in Vmax (673 vs. 702 mumol/(mg protein.min] or a decrease in Km (0.43 vs. 0.48 mM) in chronic hypercapnia as compared with controls. Metabolic acidosis also failed to increase H+-ATPase activity. These data demonstrate that the H+-ATPase of renal medulla does not display the expected increase in activity during acidosis. The role of this H+-ATPase in the adaptation to acidosis remains to be determined.     

植物新药——甘木通的生药鉴别

木文对甘水通的生药结构、药用情况、鉴别点、地理分布及繁殖等进行了报道;同时指出造成混乱的原因及找出混乱种的学名。甘本通为分布于广东和广西两省区的特有种。     

DNA拓扑异构酶Ⅰ生物学活性的比较研究

测定了3T3细胞、人和大鼠一些组织中DNA拓扑异构酶Ⅰ的活性;估计了核酸内切酶对拓扑酶Ⅰ松弛活性测定的干扰程度;发现增殖组织全细胞抽提液中酶比活高于正常分化组织,而且在异常增殖组织中酶比活的增高更为显著。     

培养的人脐静脉内皮细胞合成的蛋白聚糖

以[~(35)S]-Na_2SO_4为示踪物,观察培养的人脐静脉内皮细胞(EC)合成及分泌的蛋白聚糖(PG),经DEAE-Sephacel离子交换及Sepharose6B凝胶滤柱层析分析发现细胞层及培养液均含有三种PG单体,即硫酸乙酰肝素蛋白聚糖(HS-PG)、硫酸软骨素蛋白聚糖(CS-PG)及硫酸皮肤素蛋白聚糖(DS-PG)HS-PG又可分为大小两种,前者(HS-PG_L)位于V_o处,后者(HS-PG_s)Kd=0.53(sepharose6B);CS-PG/DS-PG分为三个峰,峰Ⅰ位于V_0处,峰Ⅱ、峰Ⅲ的Kd值分别为0.26及0.52(sepharose6B)。汇合前后细胞层及培养液中各种PG的含量不同。细胞层PG总量汇合前低于汇合后,无论是细胞层还是培养液汇合前HS-PG_L均低于汇合后,HS-PG_L与HS-PG_s比值亦为汇合前低于汇合后,而CS-PG/DS-PG含量则高于汇合后。汇合前后EC合成及分泌PG的差异与文献报道的EC损伤及正常者类似。     

转化细胞及腹水癌细胞对生长调节因子敏感性的研究

 本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。     

Role of the N-terminal region of the a chain in β1-bungarotoxin from the venom ofBungarus multicinctus (Taiwan-banded krait)

The N-terminal α-amino groups of β₁-bungarotoxin (β₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the α-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between β₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that β₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the α-amino group of the A chain was in the vicinity of substrate binding site and that the TNP α-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for β₁-Bgt.