TARGETING ACTIVATED LYMPHOCYTES WITH PHOTODYNAMIC THERAPY: SUSCEPTIBILITY OF MITOGEN-STIMULATED SPLENIC LYMPHOCYTES TO BENZOPORPHYRIN DERIVATIVE (BPD) PHOTOSENSITIZATION

Abstract— Benzoporphyrin derivative monoacid ring A (BPD), a hydrophobic chlorin-like porphyrin derivative, which fluoresces strongly at 690 nm, may have potential for both oncologic and nononcologic applications in photodynamic therapy (PDT). To study the influence of cellular characteristics on the uptake of BPD, the murine tumor cell line (P815), and in vitro and in vivo concanavalin A (Con A)-stimulated and unstimulated murine splenic lymphocytes were incubated with 2 µg/mL BPD at 37°C for 0–60 min. At various times, cells were lysed and the amount of BPD taken up by the cells was quantified by fluorescence measurements. The subsets of cells taking up BPD were analyzed using a panel of monoclonal antibodies and the Coulter XL* fluorescence-activated cell sorter. Furthermore, Con A-stimulated and unstimulated spleen cells were incubated with 0–50 ng/mL of BPD for 1 h prior to exposure to red light (7.2 J/cm²). Cell survival 24 h post-PDT was measured by the MTT assay. We found that the rapidly dividing tumor cell line and mitogen-stimulated murine T cells (mainly CD4V IL-2R⁺) took up significantly more BPD (5–10-fold) than do unstimulated splenic lymphocytes. Increased BPD uptake correlated with greater photoinactivation when these cells were exposed to light at a wavelength of 690 nm. These findings suggest that activated cells of the immune system may be a target for photoinactivation by BPD.     

PHOTOOXIDATION OF CELL MEMBRANES USING EOSIN DERIVATIVES THAT LOCATE IN LIPID OR PROTEIN TO STUDY THE ROLE OF DIFFUSIBLE INTERMEDIATES

Eosin derivatives that bind primarily to lipid or protein sites in erythrocyte membranes were studied in solution and as sensitizers of erythrocyte membranes. In 50% ethanol-water mixtures eosin maleimide (EYMA) and 5-N-hexadecanoyl amino eosin (E16) had nearly identical absorption spectra. Higher ethanol concentrations did not change peak absorbances. In the presence of neutral detergent both sensitizers had equivalent absorbance at all ethanol concentrations. In water, EYMA was more effective than E16 at bleaching RNO, probably because of E16 aggregation into micelles, while in ethanol-water mixtures E16 was slightly more effective at bleaching DPBF, indicating equivalent singlet oxygen generation when the sensitizers are in monomeric form. In water with neutral detergent, azide in the 20 microM range inhibited the majority of RNO bleaching with both sensitizers; in 50% ethanol-water mixtures azide at 1 mM showed a 50% inhibition of DPBF bleaching with both sensitizers. Iodide in the 30 mM range reduced DPBF bleaching by 50% in 50% ethanol-water mixtures. When matched for amount loaded in erythrocyte membranes these sensitizers were about equally effective at sensitizing induction of cation permeability, assayed as rate of delayed photohemolysis, while E16 was slightly more effective at sensitizing loss of cholinesterase (AchE) activity. The relation of lysis rate to load was somewhat steeper for E16 than EYMA. For both sensitizers lysis rate increased at about the 1.5 power of light dose. Deoxygenation of the reaction media with argon totally blocked detectable photomodification. Ghost membranes made from sensitizer-treated cells were effective generators of singlet oxygen, assayed by RNO bleaching. However, when mixtures of EYMA-treated and untreated cells were illuminated together, only the EYMA-treated cells showed evidence of photomodification. Azide at 5 mM slowed the initial rate of AchE loss by about 75% with E16 and EYMA. Azide partially slowed photohemolysis. Azide decreased RNO bleaching by sensitizer-treated ghosts as it did in water with detergent micelles. A deuterium oxide solvent increased photohemolysis rate with E16 by 41%, but did not increase photohemolysis rate with EYMA. Deuterium oxide had a positive, but statistically insignificant effect on loss of AchE with both sensitizers. Deuterium oxide following illumination slowed lysis sensitized by both sensitizers more than 50%. Iodide exerted a modest inhibition of photohemolysis and loss of AchE sensitized by E16, but had virtually no influence on sensitization by EYMA. The results in solution indicate that EYMA and E16 have nearly identical photochemical properties when in monome     

PROPERTIES OF IMMOBILIZED THYLAKOID MEMBRANES IN A PHOTOSYNTHETIC PHOTOELECTROCHEMICAL CELL

Abstract— Thylakoid membranes isolated from spinach were immobilized in a cross-linked albumin-glutaraldehyde matrix. Their properties in a single-compartment photoelectrochemical cell using platinum electrodes in potentiostatic mode were compared with the native material. The porous network of the immobilized tissue retained a relatively significant quantity of aqueous media which could be modified at will by aspiration of the proper media. In the presence of the acceptor potassium ferricyanide the photocurrent generation was enhanced up to a value of one hundred times higher than the maximum output of thylakoids entrapped in a polyvinyl alcohol film deposited on a Sn0₂ electrode. Immobilization in the albumin-glutaraldehyde matrix resulted in a better resistance to high pH, elevated temperatures and continuous exposure to high light intensity in potentiostatic mode (working conditions) in the electrochemical cell.     

PROTOPORPHYRIN-SENSITIZED PHOTODYNAMIC MODIFICATION OF PROTEINS IN ISOLATED HUMAN RED BLOOD CELL MEMBRANES

Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross-linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Most sensitive to this photodynamic effect are spectrin and band 2. 1, 2. 2, 2.3 and 4.1. polypeptides, which are cross-linked after very brief illumination periods, with a concomitant loss of spectrin-associated ATPase activity. Band 6 protein, representing the monomeric form of glyceraldehyde-3-phosphate dehydrogenase, is also very sensitive to protoporphyrin-induced cross-linking. The enzymatic activity decreased even faster than the amount of band 6 polypeptides, suggesting that modification(s) of the enzyme other than cross-linking, possibly by rapid photooxidation of a thiol group, may be responsible for inactivation.
Extracted and purified spectrin was cross-linked with about the same velocity as membrane-bound spectrin, reinforcing our previously drawn conclusion that membrane lipids are not involved in the cross-linking reaction. Eluted band 6 polypeptides on the other hand exhibited a relatively fast photo-oxidative modification but a much slower cross-linking to dimers and tetramers. This suggests that the membrane structure, e.g. the spectrin matrix may play an essential role in the incorporation of membrane-bound band 6 polypeptides in the high molecular weight cross-linked complex.     

电渗析提纯含氯化钠的9-氨基壬酸

用离子交换膜“Permaplex”,C-20和A-20电渗析法煶纯合氯化钠的9-氨基壬酸,当每100毫升电渗析溶液中合3.5克9-氨基壬酸及1.5克氯化钠时,优惠条件与实验结果如下: 1.中间槽溶液的pH值为7.5—7.8; 2.最大电流密度约为8毫安/厘米~2;电压为65伏; 3.阴极液——氢氧化钠的浓度为0.00135±0.0002N;阳极液——盐酸的浓度为0.0040±0.0002N; 4.电导值为5×10~(-3)欧姆~(-1)厘米~(-1)时,停止电渗析操作。 应用上述条件,用电渗析法除去9-氨基壬酸中氯化钠后,于水中重结晶两次得9-氨 基壬酸纯品,熔点186—189℃,灰分含量不大于0.1％,氯根微量,其收率约93％。     

MODULATION OF 8-METHOXYPSORALEN-DNA PHOTOADDUCT FORMATION BY CELL DIFFERENTIATION, MITOGENIC STIMULATION AND PHORBOL ESTER EXPOSURE IN MURINE T LYMPHOCYTES

Abstract The effects of cell differentiation and mitogen and phorbol ester stimulation on the formation of 8-methoxypsoralen (8-MOP)-DNA photoadducts in murine T lymphocytes were examined using ³H-8-MOP. While there were no significant differences in 8-MOP photoadduct formation among BALB/c thymocytes, splenocytes, splenic T cells and MRL/1pr lymph node cells, BALB/c bone marrow cells showed fewer photoadducts than did the lymphocytes. This suggested that proliferating progenitor cells may be resistant to 8-MOP photoadduct formation. Incubation of purified splenic T cells with lectin mitogens for 2 h or with phorbol 12-myristate 13-acetate (PMA) for 2–43 h resulted in reduction of 8-MOP photoadduct formation in the DNA, whereas 64 h cultivation with these agents augmented the photoadduct formation. The reduction of photoadduct formation induced by phytohemagglutinin was restored by the further addition of a protein kinase C (PKC) inhibitor, H-7, to the culture. Thus, it is assumed that the reduction of adduct formation evoked by mitogens and PMA is mediated in part by the activation of PKC in the cells. On the other hand, the augmentation of the adduct formation induced by the longer-period cultures with mitogens and PMA appeared to be caused by down-regulation of PKC. The present study showed that the stimulatory signals in which PKC is presumably involved affect the ability of cells to form 8-MOP-DNA photoadducts.     

EFFECTS OF in vitro EXPOSURE TO ULTRAVIOLET RADIATION ON THE FUNCTIONAL ACTIVITY OF LYMPHOCYTES, WITH EMPHASIS ON SUSCEPTIBILITY OF DIFFERENT SPECIES

Abstract In this study lymphocytes from blood and/or spleen of different species (rat, mouse, human) were exposed to different doses of ultraviolet radiation (UVR). The functional activity of these lymphocytes was determined using assays for mitogen proliferation and the mixed lymphocyte response (MLR). These experiments demonstrated that in vitro exposure to UVR causes a dose-dependent decrease of the MLR activity of the irradiated lymphocytes. Viability of lymphocytes and mitogen proliferation responses were also decreased by UVR exposure but less severe in comparison to the MLR. Lymphocytes of rats seem to be more sensitive to UVR as compared to lymphocytes of mice and humans.     

SURFACE MULTI-FUNCTIONALIZATION OF POLY(LACTIC ACID) NANOPARTICLES AND C6 GLIOMA CELL TARGETING in vivo

Polysaccharide coated PLA nanoparticles bearing aldehyde groups were prepared by dialysis of DMSO solution of cholesterol hydrophobic-modified dextran polyaldehyde and PLA against water.The average diameter of the nanoparticles was about 160 nm,and the size distribution was nearly homogenous.The nanoparticles were functionalized simultaneously with CD71 and EGFR antibody through the Schiff's base reaction,and then radiolabeled with ~(99m)Tc.After perfused the radiolabeled nanoparticles into tumor-bearing...     

MECHANISMS OF CELL KILLING IN PHOTODYNAMIC THERAPY USING A NOVEL in vivo DRUG/in vitro LIGHT CULTURE SYSTEM

Photodynamic therapy of certain neoplasms has emerged as a promising form of cancer treatment. This type of therapy involves the exogenous administration of a photosensitizer with subsequent exposure to light. The ensuing photochemical reaction results in destruction of the tumor. Whether tumor cells are destroyed directly by the photodynamic treatment or indirectly as a result of destruction of the tumor microvascular bed is unknown. To address this question, methods were adapted to test whether combinations of a photosensitizer and light resulted in direct cell killing of precision cut tissue slices placed in culture. The major advantages of this culture system are that photosensitizers are administered in vivo, tissue slices produced in minutes, placed in culture medium, and irradiated in vitro. Any resulting cellular destruction occurs in the absence of a functioning vascular system and indicates that photodynamic therapy acts through a direct cell killing mechanism. Tissue slice viability was monitored by two standard methods: assay for intracellular potassium and morphological examination at the electron microscopic level. The effects of hematoporphyrin derivative and light were examined on tissue slices produced from a prostate adenocarcinoma transplanted into male Copenhagen rats. The data indicate that direct killing of tumor slices occurs and is dependent on the irradiation protocol used.     

PREPARATION OF EVOH MICROPOROUS MEMBRANES via THERMALLY INDUCED PHASE SEPARATION USING BINARY SOLVENTS

Microporous ethylene-vinyl alcohol copolymer （EVOH） flat membranes and hollow-fiber membranes with 38 mol% ethylene content were prepared via thermally induced phase separation （TIPS） using the mixture of 1,4-butanediol and poly（ethylene glycol）（PEG400） as diluents. Effects of the ratio of 1,4-butanediol to PEG400 on the phase diagrams, phase separation mechanism and membrane morphology were studied by small angle light scattering （SALS） measurements, differential scanning calorimetry （DSC）, and scanning ele~：tron microscopy （SEM）. It was found that by varying the composition of the binary solvent, the phase diagrams and membrane morphology can be controlled successfully. Moreover, the phase diagrams showed that broader regions of Liquid-Liquid （L-L） phase separation were obtained, as well as closer distances between L-L phase separation lines and Solid-Liquid （S-L） phase separation lines, Interconnected structures observed both in the flat membrane and hollow fiber membrane consist with the above results.     

cis-UROCANIC ACID FAILED TO AFFECT in vitro HUMAN LANGERHANS CELL ALLOSTIMULATORY FUNCTION

Abstract— -Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290–320 nm)-absorbing component of the skin. Trans-UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis -UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell-lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5–400 μg/mL) of purified cis-UCA or (rara-UCA did not modify the T-cell response supported by enriched LC (eLC: 8–25% LC) as well as purified LC (pLC: 70–90% LC) suspensions. Because cis-UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T-cell proliferation induced by UVB-irradiated LC. The UVB exposure of eLC or pLC to 100 J/m² significantly inhibited the capacity of both suspensions to mount a T-cell response. However, addition of cis- UCA did not potentiate this UVB-induced immunosuppression. The eLC or pLC were then incubated with cis-UCA for 18 h at 37°C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis -UCA did not alter human LC function. In conclusion, these results strongly suggest that cis-UCA has no direct effect on human LC antigen-presenting function.     

PORPHYRIN PHOTOSENSITIZATION OF PROTEINS IN CELL MEMBRANES AS STUDIED BY SPIN-LABELLING AND BY QUANTIFICATION OF DTNB-REACTIVE SH-GROUPS

Abstract— Human cells of the line NHIK 3025 were exposed to hematoporphyrin derivative (Hpd) and light and analysed with respect to; (i) the mobility of membrane proteins as determined by electron spin resonance measurements of a protein-bound spin label, (ii) fluorescence excitation spectra, (iii) relative number of DTNB-reactive SH-groups on their surface and in sonicated cell homogenates, (iv) survival, and (v) morphologic appearance as seen by ordinary phase contrast microscopy. A significant fraction of the porphyrins bound to the outer cell membrane was in close contact with proteins. 5,5'-Dithiobis-2-nitrobenzoic acid reactive SH-groups on the outer cell membrane were very sensitive to the treatment with Hpd + light and were degraded according to non-exponential kinetics. When the cells were irradiated after spin labelling, the labelled proteins became less mobile during the irradiation, indicating protein cross linking. Irradiation before spinlabelling resulted in a selective degradation of low-mobility proteins.     

OXIDATION OF BENZOIN TO BENZIL USING BURGESS REAGENT

ABSTRACT

Synthetic utility of Burgess Reagent for the mild and efficient oxidation of benzoins to benzils is discussed.     

SENSITIVITY OF THE ELECTRON TRANSPORT CHAIN OF PIGMENTED AND NON-PIGMENTED SARCINA MEMBRANES TO PHOTODYNAMIC ACTION

Abstract— With malate as substrate, the respiratory system of Sarcina lutea is subject to photodynamic action when toluidine blue is added as a photosensitiser. Both whole cells and isolated cell membranes show similar photoinactivation. Cell and membrane suspensions of a carotenoid-containing strain are significantly less sensitive than those from a non-pigmented mutant prepared from the pigmented strain. Addition of histidine also provides photoprotection. Attempts to locate the site of photoinactivation within the respiratory system lead to the conclusion that several sites are affected. Short periods of illumination do not result in permanent damage since activity is recovered in these suspensions when incubated in the dark subsequent to illumination. The significance of these results and the possible mechanisms involved are discussed.     

SEPARATION OF PROPANE FROM PROPANE/NITROGEN MIXTURES USING PDMS COMPOSITE MEMBRANES BY VAPOR PERMEATION

This study deals with polydimethylsiloxane(PDMS)/polyvinylidene fluoride(PVDF) composite membranes for propane separation from propane/nitrogen mixtures,which is relevant to the recovery of propane in petroleum and chemical industry.The surface and cross-section morphology of PDMS/PVDF composite membranes was observed by scanning electron microscope(SEM).The surface morphology of PDMS/PVDF composite membranes is very dense.There are three layers,the thin dense top layer,finger-like porous middle layer an...     

PHOTOTACTIC RESPONSE OF CHLAMYDOMON AS TO FLASHES OF LIGHT-I. RESPONSE OF CELL POPULATIONS

Abstract— Chlamydomonas reinhardi responds phototactically to a single, very short flash of blue light (6-4 μs). Net oriented response of a cell population is monitored photometrically, using the "population system" of Feinleib and Curry (1967). A single high-intensity flash elicits a small, but definite net movement away from the stimulus source. Repetitive flashing at low frequency (between 8 and 60 flashes per min) and at the same intensity elicits a prolonged response in the same direction. Net phototactic response to single or repetitive flashes varies with stimulus intensity in the same way as does response to continuous light (Feinleib and Curry, 1971b); response is positive at low intensity and negative at high intensity. These data indicate that at least some cells become oriented in response to a short flash. The occurrence of such a response has implications for the mechanism of phototactic orientation. If almost all the cells responded, one would assume that Chlamydomonas perceives light direction instantaneously by detecting an absorption gradient within the cell. Unequivocal interpretation of the short-flash response requires examination of the behavior of individual cells.     

EXCIMER FORMATION OF N-(I-PYRENESULFONYL)-DIPALMITOYL-L-α- PHOSPHATIDYLETHANOLAMINE AT THE LIPID-WATER INTERFACE OF FAT CELL PLASMA MEMBRANES

Abstract— The mechanism of excimer formation of N-(l-pyrenesulfonyl)-dipalmitoyl-L-α-phosphatidylethanolamine incorporated into fat cell plasma membranes was studied by means of steady-state and pulse fluorometry. It was found that the pyrenesulfonyl group at the lipid-water interface of the membrane formed an appreciable amount of ground-state dimers and that the excimer of the pyrenesulfonyl group was formed through two different processes: (i) the collisional interaction of an excited monomer with a ground-state monomer, and (ii) the direct excitation of a ground-state dimer (approximate ratio of the two processes, 1:1). Analysis of fluorescence decay curves revealed that the detailed mechanism of excimer formation by process (i) is essentially similar to the mechanism established for the reaction in organic solvents but not to that in a highly viscous medium such as the hydrocarbon core of the membrane.     

具有荧光探针的靶向肽/聚(醚-酰胺)制备及其肝癌细胞靶向性能研究

通过端氨基聚乙二醇PEG(Ⅰ)与二亚乙基三胺五乙酸二酐(DTPAA)开环合成新型端氨基聚(醚-酰胺)(PEG/DTPA)共聚物造影剂配体(Ⅱ)(Step1);Ⅱ的端氨基与偶联剂3-马来酰亚胺苯甲酸-N-琥珀酰亚胺酯(MBS)的活化端COOH反应,生成偶联剂/聚(醚-酰胺)MB/PEG/DTPA(Ⅲ′)(Step2);再通过Ⅲ′中MBS的CC双键与肝癌细胞靶向黏附肽FAM-AGKGTPSLETTPC-(SH)-COOH(FAM-13)上的巯基SH发生Michael加成反应(Step3),合成含有荧光探针FAM(5-carboxyfluorescein)的肝癌靶向肽/聚(醚-酰胺)(FAM-13/PEG/DTPA,Ⅲ).用1H-NMR和13C-NMR等方法对共聚物进行表征.Ⅲ对正常肝细胞L-02几乎观察不到荧光现象,而对肝癌细胞BEL-7404则有很强的黄绿色荧光,Ⅲ对肝癌细胞有很强的靶向性.大分子配体Ⅲ可望用于制备大分子造影剂及靶向载体负载药物.     

In vitro DEMONSTRATION OF A FUNDAMENTAL DIFFERENCE IN THE PROLIFERATION OF MURINE and HUMAN BONE MARROW and LYMPHOCYTES FOLLOWING ULTRAVIOLET IRRADIATION: RELEVANCE TO BONE MARROW TRANSPLANTATION

Exposure of rodent allogeneic donor marrow and splenocyte grafts to ultraviolet radiation (UVR) has been shown to permit durable engraftment at doses that abolish graft-versus-host disease (GVHD) and graft rejection. We have compared both murine and human alloreactive and mitogen-induced lymphoid responses and bone marrow proliferation in mixed lymphocyte culture (MLC), phy-tohemagglutinin (PHA)-induced proliferation and colony-forming unit-granulocyte/macrophage (CFU-GM) assays using germicidal UVC (200–290nm), broadband and narrowband UVB (290–320nm) and UVA (320^100 nm) sources. Our data show a wavelength and dose-dependent reduction in lymphoid proliferation in the mouse with CFU-GM survival of50–75% of control at doses required to abolish allogeneic lymphocyte responses for all lamps. In contrast, human lymphocyte responses are more resistant to UVC with CFU-GM proliferation reduced to zero when allostimulation is abolished. Mito-gen-induced lymphoid responses show a similar wavelength-dependent sensitivity. Abolition of response in MLC using UV-irradiated stimulator cells was less sensitive than proliferation with UV-irradiated responder cells at all wavelengths in both species. With all sources, murine CFU-GM proliferation is less susceptible to UVR than human marrow at doses required to abolish lymphoid responses.     

DEVELOPMENT OF AN in vitro SYSTEM FOR THE ANALYSIS OF ULTRAVIOLET RADIATION-INDUCED SUPPRESSION OF NATURAL KILLER CELL ACTIVITY

Previous studies have shown that natural killer (NK) cell activity was suppressed in volunteer subjects exposed to ultraviolet radiation (UVR) from solarium lamps. The present studies were carried out to determine that spectrum of UVR responsible for suppression of NK activity and to develop in vitro methods to analyze the effectivenes of sunscreen agents in prevention of UVR-mediated suppression of NK activity and other aspects of immune function. UVR from a xenon are lamp source was used to irradiate peripheral blood lymphocytes (PBL) in wells of tissue culture flasks, and transmission interference filters were used to eliminate UVR of particular wavelengths. The results indicated that UVR from this source inhibited NK activity of PBL in a dose-dependent manner with a 50% inhibitory dose of 5.5 mJ/cm² when unfiltered and 29.6 mJ/cm² when diluted through cellulose acetate, which gave a UV spectrum similar to that in solar radiation. Equivalent suppression of NK activity was mediated by UV-A (UVR > 315 nm) at dose levels of 4.2 J/cm², which was approximately 140 times greater than the amount of UV-B (UVR > 315 nm) needed to suppress NK activity. Similar dose-response curves were seen for inhibition of mitogenic responses to phytohemagglutinin except that the latter appeared less sensitive than NK to inhibition by UV-A. These studies suggest that whe the greater proportion of UV-A in solar radiation adn its greater penetration into skin is taken into account, UV-A may have equivalent or greater direct immunosuppressive effects than UV-B. The mechanisms of their immunosuppressive effects may, however, differ. The in vitro system described here would appear to provide a simple test system for further analysis of UVR-indued imunosuppression.