Control of photoinduced electron transfer from zinc-porphyrin to methyl viologen by supramolecular formation between monoclonal antibody and zinc-porphyrin.

Photoinduced electron transfer from tetrakis(4-carboxy-phenyl porphyrin)-zinc complex (Zn-TCPP) to an acceptor molecule (methyl viologen; MV2+) has been found to be controlled by the complex formation of monoclonal antibody 03-1 for the porphyrin (TCPP) and Zn-TCPP. Although there are no ground-state interactions between Zn-TCPP and MV2+ for a 2:1 complex of antibody 03-1 and Zn-TCPP, the fluorescence of Zn-TCPP is quenched by the addition of MV2+. The Stern-Volmer plots and emission lifetime studies show that there is a long-range electron transfer through the antibody 03-1.

Functional activation of human neutrophils by recombinant monocyte-derived neutrophil chemotactic factor/IL-8.

Monocyte-derived neutrophil chemotactic factor (MDNCF)/IL-8, a novel cytokine, distinct from IL-1 and TNF was recently purified and cloned. This study was performed to investigate the biologic effect of recombinant MDNCF/IL-8 on human polymorphonuclear neutrophils (PMN) by assessment of their growth inhibitory activity against Candida albicans. The chemoattractant, FMLP was used as a positive control. We demonstrated that MDNCF/IL-8, similar to FMLP, effectively enhanced PMN-mediated anti-Candida activity. MDNCF/IL-8, from 1.0 to 1000 ng/mol, enhanced PMN-mediated anti-Candida activity, whereas FMLP was effective from 10(-10) to 10(-7) M. The optimal dose of MDNCF/IL-8 for PMN stimulation was 10 ng/ml which equalled the optimal chemoattractant dose. MDNCF/IL-8 itself, like FMLP, had no direct effect on Candida growth at any concentration and it stimulated antifungal activity only in PMN but not in monocytes. Interestingly, MDNCF/IL-8 failed to stimulate directly the production of superoxide from PMN or prime the respiratory burst of PMN exposed to FMLP. However, MDNCF/IL-8 was capable of releasing azurophilic enzymes from cytochalasin B-treated PMN into the extracellular space. Enhancement of PMN anti-Candida activity and release of azurophilic enzymes from PMN by MDNCF/IL-8 were inhibited in the presence of colchicine, which is a known inhibitor of degranulation. These results suggest that MDNCF/IL-8 induced antifungal action of PMN via oxygen-independent pathways. Furthermore, MDNCF/IL-8 induction of anti-Candida action by PMN was inhibited by pretreatment with Bordetella pertussis toxin, suggesting that enhancement of PMN antifungal activity by MDNCF/IL-8, as well as by FMLP, may be mediated by a GTP-binding protein.

Characterization of lymphocytes in a patient with Wiskott-Aldrich syndrome: studies by fluorescence polarization.

Lymphocytes from a patient with Wiskott-Aldrich syndrome (WAS) were employed for a study of the intracellular viscosity and fluorescein permeability through the cell membrane by a fluorescence polarization spectrofluorometer, which was designed to calculate polarization value and permeable fluorescein intensity automatically. Fluorescein diacetate (FDA) was used as the indicator probe. The fluorogenic substrate is taken up by viable cells and converted to a fluorescent molecule, fluorescein, by intracellular esterase, where upon the fluorescein easily effluxes through the cell membrane. The response to stimulation with phytohemagglutinin (PHA) for 45 min led to a decreased polarization value (p-value) as compared to lymphocytes of healthy donors, and the fluorescein efflux through the cell membrane was greater than that of healthy donors. Fluorescein efflux from lymphocytes in the patient during 48 and 72 h incubation with or without PHA was markedly increased. In healthy donors, the degree of fluorescein permeability was not increased during the culture. These results indicate that intracellular viscosity of lymphocytes is altered in initial mitogenic stimulation, but that there was some abnormality in the fluorescein permeability properties through the cell membrane of lymphocytes in a patient with WAS.

Human polymorphonuclear leukocyte activating factor isolated from Aleurites Fordii Hemsl. (Euphorbiaceae) seed.

A human polymorphonuclear leukocyte (PMN) activating factor (AFH-S) was isolated from a tropical plant seed, Aleurites Fordii Hemsl. (AFH) in PBS. It is found that the AFH-S stimulate human PMNs and induced superoxide generation and chemotaxis. Superoxide generation was affected by the extracellular calcium ion or pretreatment with H-7 (PK-C inhibitor), but not by mepacrine (PLA2 inhibitor) or pertussis toxin (islet-activating protein: IAP). Furthermore, a lag time exists dose-dependently. In addition the cytosolic calcium was not increased by the stimulation with AFH-S. Thus, the receptor for AFH-S was suggested to be independent from Ni-like protein, PI-response, or PLA2-activation, and stimulate PMNs through activation of PK-C.

Functional heterogeneity of human monocytes--with a special reference to flow cytometric assays.

A large number of human mononuclear cells were simultaneously separated into fractions enriched in B cells, T cells, large granular lymphocytes (LGL) and monocytes by centrifugal elutriation. Lymphocyte populations were analyzed using monoclonal antibodies. In particular, highly enriched natural killer cells, Leu7+ cells, were collected in the intermediate fractions. Monocytes, which were identified as esterase positive cells, and Leu M3 cells were collected at higher counterflow rates and in the final fraction. The purity of monocytes in the final fraction was 81%. The oxidative metabolic activity (H2O2 production) and non-specific esterase activity of individual monocytes was estimated in the analysis of functional heterogeneity of monocytes using flow cytometry. 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescein diacetate (FDA) were used as indicators in the measurement of H2O2 generation and esterase activity. Intracellular generation of a fluorescence product (H2O2 Production; average percentage of fluorescence positive cells) of monocytes in the stimulation of phorbol myristate acetate (PMA, 100 ng/ml) was greater in larger than smaller cells. H2O2 production gradually increased from 6% and 25-38% and 60% in the intermediate and final fractions respectively. Furthermore, the average fluorescence intensity of the large monocyte population in the final fraction was 1.13-1.31 fold more active than that of the smaller cells. Thus, the functional heterogeneity of human monocytes was further confirmed in the assays of H2O2 production exposed to PMA and FDA hydrolysis using flow cytometry. Furthermore, the CCE system can isolate lymphocyte subsets and LGL.

Differentiation-associated carbohydrate chain on human hematopoietic cells recognized by Clerodendron trichotomum lectin.

In the screening of hematopoietic cell line cell aggregations, the extract of Clerodendron trichotomum seed was found to aggregate K-562 and KG-1 specifically. In the flow cytometric analysis using FITC-conjugated purified CTL, it was confirmed that CTL recognizes the specific carbohydrate(s) which seem to appear only in the early stages of differentiation of myeloid (KG-1) and erythroid (K-562) cell line cells and erythrocytes. The CTL binding to K-562 cells was decreased by TPA treatment which is known to induce retrodifferentiation of K-562. It is also found that this carbohydrate(s) were shaded with NANA on the differentiated cells. In the erythrocyte, CTL receptor was partially shaded by NANA.

Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization.

The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes. Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation. Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively. Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate. The P-values were calculated for high, intermediate, and low density fractions as 0.192 +/- 0.021, 0.172 +/- 0.019, and 0.147 +/- 0.012, respectively. Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells. Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells. The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.