Effect of protein-bound uraemic toxins on the thermodynamic characteristics of human albumin

The ability of albumin to bind drugs and other lipophilic organic acids is decreased in chronic renal failure by the accumulation of albumin-bound uraemic toxins such as hippuric acid, indoxyl sulphate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). This furan acid is the most highly bound and is not removed by haemodialysis. The inhibitory effects of these three uraemic toxins on the interaction of three marker ligands sodium octanoate (for medium chain fatty acids), salicylic acid and phenol red (bilirubin site/site I) with albumin have been investigated by differential scanning microcalorimetry and flow microcalorimetry. CMPF was the most potent inhibitor and its binding site coincided with that of bilirubin (site I). Indoxyl sulphate binds to the site for medium-chain fatty acids and tryptophan (site II) and hippuric acid, the weakest inhibitor, inhibited binding to the salicylic acid site.     

细胞凋亡信息分子Fas、FasL和NF—kB在氟化物作用后破骨样细胞凋亡过程中的表达

目的：研究氟化物作用后破骨细胞样细胞凋亡过程中细胞凋亡配体Fas、FasL（Fas Ligand)和核因子NF－kB的表达变化,方法：在培养基中分别加入不同浓度的氟化钠培养破骨样细胞,以免疫组织化学染方法观察细胞的Fas、FasL和NF－kB表达,结果：Fas、FasL在破骨样细胞表达量随培养基中氟化钠浓度增高而增高,NF－kB的表达量随培养基中氟化钠浓度增高而降低,两者均具有剂量依赖关系,结论：在氟化钠所致的破骨细胞凋亡过程中,Fas、FasL的表达氟化物浓度增高而加强,而核因子NF－kB的表达随氟化物浓度增高而减弱,两者都具有剂量依赖关系。     

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.     

Suppression of Postmitochondrial Signaling and Delayed Response to UV-induced Nuclear Apoptosis in HeLa Cells

Activation of postmitochondrial pathways by UV irradiation was examined using mouse lymphoma 3SB and human leukemic Jurkat cells and two human carcinoma cell lines (HeLa and MCF-7). Exposure of 3SB and Jurkat cells resulted in large amounts of cytochrome c and apoptosis-inducing factor (AIF) being released into the cytosol, and a clear laddering pattern of DNA fragments was observed within 3 h of incubation after irradiation. Simultaneously, activation of caspase-9 and its downstream caspases was detected. HeLa and MCF-7 cells also showed extensive release of mitochondrial factors and caspase-9 activation at 4 to 6 h after exposure, but apoptotic nuclear changes appeared much later. Compared with 3SB and Jurkat cells, these carcinoma cell lines exhibited reduced activation of caspase-9-like proteolytic activity by UV radiation, and levels of caspase-3-like activity in HeLa cells were extremely low, similar to those in caspase-3-deficient MCF-7 cells. These results suggest that the delayed response to UV-induced nuclear apoptosis in HeLa cells is due to a reduced activation of the caspase cascade downstream of cytochrome c release and suppression of caspase-3 activity.     

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.     

Time-course of translocation and dicentric frequencies in a radiation accident case

PURPOSE: To assess the persistence of exchange aberrations measured by FISH chromosome painting after accidental radiation exposure. MATERIALS AND METHODS: Chromosome analyses were carried out in peripheral lymphocytes of a 13-year-old boy exposed to protracted low dose-rate whole-body and short-time partial-body irradiation from a radiation accident in Estonia in 1994. Up to November 1998, the frequencies of translocations and dicentrics were periodically measured using FISH chromosome painting of the target chromosomes 1, 4 and 12, with a simultaneous pancentromeric probe. RESULTS: For the yields of dicentrics, an expected rapid temporal decline was found with a half-time of 14.2+/-1.9 months. The yields of reciprocal translocations also revealed a gradual but significant reduction with a half-time of 51.7+/-12.7 months. CONCLUSION: An unchanged temporal persistence of so-called stable translocations cannot be assumed. Any significant reduction of this aberration type with time obviously limits the application of FISH-based translocation measurements for reliable long-term biodosimetry after combined protracted whole-body and partial-body radiation exposure.     

A quantitative assessment of changes in the dermal fibroblast population of pig skin after single doses of X-rays

Investigations were carried out into the time- and dose-related changes in the density of fibroblasts in the dermis of irradiated pig skin. The time course of these changes in the density of fibroblast nuclei in the reticular dermis was studied from 6 to 104 weeks after irradiation with a single dose of 15.4 Gy of X-rays. The largest decrease in the number of fibroblasts occurred between 12 weeks and 26 weeks after irradiation; after this time there was only a slight fall in the fibroblast number until 104 weeks when the observations ceased. At 26 weeks and later times after irradiation the reduction in the density of fibroblast nuclei in the reticular dermis was dose-dependent for single doses in the range 8.0-20.7 Gy. The dose-response curve had an initial shoulder, after which the fall in the fibroblast nuclear density was linearly related to dose. Data obtained at other times, between 26 weeks and 104 weeks after irradiation, could be fitted by the same dose-response curve. The fall in the counts of fibroblast nuclei was compared with earlier studies in pig skin. The loss of fibroblasts occurred after an initial reduction in blood flow in the pig skin but was concomitant with the general reduction in dermal thickness.     

An autoradiographic study of the intrarenal localisation and retention of cisplatin,iproplatin and paraplatin

Summary The intrarenal localisation of platinum following the intravenous administration of platinum-195m-labelled cisplatin, iproplatin and paraplatin was studied using autoradiography. Following injection of cisplatin, platinum was distributed throughout the kidney even up to 14 days after treatment. In the case of iproplatin and paraplatin rapid platinum clearance was noted from the glomeruli, blood vessels and renal medulla within 2 h of administration. Relative cortical and medullary platinum radionuclide concentrations for all three agents were determined by Chalkley grid analysis. This showed greater relative concentrations of platinum in the cortex at increasing times following iproplatin and paraplatin compared with cisplatin. It is suggested that the lower renal toxicity of iproplatin and paraplatin than of cisplatin may be due to reduced platinum retention within the pars recta.