Sites of photosensitization by protoporphyrin and tin protoporphyrin in leukemia L1210 cells

Studies with protoporphyrin (PP) and tin protoporphyrin (SnPP) were carried out to assess the effects of tin insertion on the sites of dye localization. Fluorescence emission spectra and studies on the sites of photodamage were consistent with a concentration of PP at membrane loci. In contrast, SnPP photodamage involved an intracellular site.     

The anti-neoplastic activity of ethylamine-carboxyborane and triphenylphosphine-carboxyborane in L-1210 lymphoid leukemia cells

Studies on the mode of action of two boroncontaining anti-neoplastic agents, ethylamine-carboxyborane and triphenylphosphine-carboxyborane, are reported. The major site of inhibition was in the pyrimidine de nove synthetic pathway at orotidine monophosphate decarboxylase activity. Additional sites which may facilitate the inhibition of cell growth were IMP dehydrogenase, thymidine kinase, TMP kinase and TDP kinase, m-RNA, r-RNA and t-RNA polymerase activities as well as topoisomerase II activity. The reduction in enzyme activities led to sufficient reduction of d(NTP) levels to suppress DNA synthesis and cell growth. DNA strand scission was evident in the presence of drug. Multiple modes of action are common with amine-carboxyboranes. Acute toxicity studies in mice showed that both agents were safe in their therapeutic range based on organ weights, histological tissue sections, clinical chemistry values and hematopoietic parameters.     

LIMITED CELL-CYCLE DEPENDENCE OF THE MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF L1210 LEUKEMIA CELLS

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.     

NanoLC-ESI MS/MS对重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白的准确鉴定

重组人Ⅱ型肿瘤坏死因子受体一抗体融合蛋白(商品名益塞普)是一种用于治疗中度及重度活动性类风湿关节炎的重组蛋白药物.该药由肿瘤坏死因子受体和人IgG1的FC片段融合而成.国内的药品报批时,没有明确规定必须为人IgG1的FC片段.但该药品出口到国际市场时,要求必须确定为人IgG1的FC片段.人IgG共有4个亚型,序列高度同源.为准确鉴定人IgG1,本实验应用超高效纳升液相色谱一高解析离子淌度质谱仪HDMS进行了分析,结果表明重组人Ⅱ型肿瘤坏死因子受体一抗体融合蛋白一级结构正确,FC片段确实为人IgG1,符合出口国际市场的标准.     

PHOTOTOXIC EFFECTS OF FOUR PSORALENS ON L1210 CELLS. THE CORRELATION WITH DNA INTERSTRAND CROSS-LINKING

L1210 mouse leukaemia cells were treated with psoralen [S-methoxy-(XMOP), 4,5′,8-trimethyI-(TMP), 4′-hydroxymethyl-4,5′,8-trimethyl-(HMT) or 4′-amino- methyl-4,5′,8-trimethylpsoralen (AMT)] in combination with long wavelength ultraviolct irradiation (Λ～ 365 nm). In order to investigate the relative photobiological activities of the psoralens, cell viability and DNA-synthesis activity as well as psoralen-DNA photoaddition and DNA interstrand cross-linking were measured after the treatment. In all assays the activity ranking order was found to he: TMP > HMT > AM7 > 8MOP. Furthermore, a direct correlation between phototoxicity, psoralen induced DNA interstrand cross-links and inhibition of DNA synthesis was indicated. Finally, psoralen uptake by the cells appears to be an important determinant for phototoxicity, whereas their DNA photoreactivity does not.     

Metastable State of the Fc Fragment

The metastable state appearing at neutral pH in the Fc fragment after its brief incubation at pH 2.5 was studied using scanning microcalorimetry, high-flow sedimentation and fluorescence polarization. It was shown that the metastable state of the Fc fragment is characterised by smaller melting enthalpy of C_H2 domains as compared with that of the stable state which is explained by reduced interaction of C_H2 and C_H3domains. As seen from the decrease in the sedimentation coefficient, this leads to diminution of compactness of the Fc fragment molecule. The intramolecular motility of C_H2 domains increases judging by the fluorescence polarization data. This revised version was published online in July 2006 with corrections to the Cover Date.     

NUCLEAR FACTOR κB BINDING ACTIVITY IN MOUSE L1210 CELLS FOLLOWING PHOTOFRIN II-MEDIATED PHOTOSENSITIZATION

Clinical photodynamic therapy (PDT) uses the photosensitizer photofnn II to produce singlet molecular oxygen and other reactive oxygen intermediates for localized tumor tissue cytotoxicity. In this report, we show that PDT enhances the DNA binding activity of nuclear factor kappa B (NFkB), a transacti vator of cytokine gene expression. Photosensitization following a 16 h incubation of photofrin II induced NFkB binding activity in mouse leukemia L1210 cells 10-fold above that observed in exponentially growing cultures. Serum starvation, as well as drug-alone and light-alone controls, elevated basal NF k B binding activity two- to three-fold. Upstream stimulatory factor binding activity was not modulated by any of the cell treatments and was used to standardize gel mobility shift data. This study identifies porphynn-mediated PDT as an inducer of NF k B binding activity, extending recent findings that NF k B activation is a general response to oxidative stress.     

Chromatographic separation and molecular modelling of triazines with respect to their inhibition of the growth of L1210/R71 cells.

The potential anti-cancer activity of triazines was characterized by the inhibition of the growth of L1210/R71 cells. The retention times for fifteen triazine derivatives were measured by high-performance liquid chromatography on octyl silica and silica gel columns. The slope and intercept values of the plot of the logarithmic capacity factor versus acetonitrile concentration were calculated from the reversed-phase retention measurements. The adsorption properties of the compounds were characterized by the retention data obtained on silica gel columns using high and low concentrations of ammonium salts in the hydro-organic mobile phase. The non-polar, non-polar unsaturated and polar surface areas, the surface energies, the dipole moments and the Van der Waals radii of the molecules were calculated from their chemical structures after energy minimization on the basis of molecular mechanics. Correlation analysis of these parameters showed that the inhibitory effect is dependent on the polar and non-polar surface areas of the molecules. The reversed-phase slope showed a significant correlation with the difference between the accessible and the total non-polar surface areas of the compounds, whereas the intercept values correlated with the non-polar accessible surface area. The adsorption properties of the triazines on silica gel cannot be described by the molecular parameters investigated here.     

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.     

A synthetic mimic of human Fc receptors: defined chemical modification of cell surfaces enables efficient endocytic uptake of human immunoglobulin-G

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.