Nobuto Yamamoto

PHOTODYNAMIC SENSITIZERS FROM CHLOROPHYLL:PURPURIN–18 AND CHLORINp6

Abstract— Two easily prepared derivatives of chlorophyll,purpurin–18 and chlorin p6, are potent sensitizers of cell killing by low‐intensity red light. The internal anhydride group inpurpurin–18 provides the potential of covalently linking in one step the chlorin to cell targeting agents such as antibodies.

Activation of peritoneal macrophages by lysophosphatidylcholine

Lysophosphatidylcholine (lyso-PC), a product of inflammation induced by infectious and other agents, is able to stimulate mouse peritoneal macrophages to ingest target cells coated with IgG but not IgM regardless of the presence of complement. In vitro treatment of mouse resident peritoneal macrophages (adherent cells) alone with lyso-PC stimulated spreading activity but did not enhance ingestion activity of macrophages. However, when mixed cultures of adherent and nonadherent (lymphocytes) cells were treated with lyso-PC, macrophage ingestion activity of IgG-coated target cells (i.e., via Fc-mediated ingestion) was markedly enhanced. Analysis of lyso-PC activation process of macrophages for ingestion activity suggests that nonadherent (lymphocytes) cells are required for the induction of the manifestation of ingestion capacity. This requirement was also met by addition of untreated nonadherent cells to treated adherent cells. Thus, the activation mechanism of macrophages by lyso-PC for ingestion requires contribution of lymphocytes to promote enhanced ingestion activity. Since lyso-PC is a metabolite of a representative membrane phospholipid, we propose that lyso-PC and other lysophospholipids are mediators for activation of macrophages regardless of the type of inflammation-causative agent.

Activation of macrophages by ether analogues of lysophospholipids.

SummaryInflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-α-lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.

Effects of inflammation products on immune systems

SummaryMicrobial infection causes inflammation which stimulates macrophage functions. One of the inflammatory products, lysophosphatidylcholine (lyso-Pc), can stimulate macrophage activities. Treatment of mice with lyso-Pc enhanced spreading and ingestion activities of peritoneal macrophages. In vitro treatment of macrophages with lyso-Pc greatly enhanced spreading but not ingestion activities. However, incubation of a mixture of adherent and nonadherent cells with lyso-Pc produced a markedly enhanced ingestion activity of macrophages, implying the contribution of nonadherent cells to the stimulation of macrophages. Time course studies of the stimulation of these macrophages showed that spreading activity is stimulated immediately, even 30 min, after their contact with lyso-Pc while induction of ingestion activity requires a latent period of about 5 h. When the specificity of the macrophage receptors for ingestion was analyzed using defined immunoglobulins (i.e., IgG and IgM) with or without complement, lyso-Pc-activated macrophages efficiently ingested IgG-coated sheep erythrocytes independent of complement. However, macrophages of the same lyso-Pc-treated mice did not ingest erythrocytes coated with IgM and complement. These observations suggest that lyso-Pc-stimulated macrophages ingest the targets via Fc-receptors but not C3b receptors.

Contribution of Lysophosphatidylcholine-Treated Nonadherent Cells to Mechanism of Macrophage Activation

Abstract Lysophosphatidylcholine (lyso-PC), a product of inflammation, stimulates (in vivo) mouse peritoneal macrophages to ingest target cells via Fc receptors. In vitro treatment of macrophages with lyso-Pc was unable to enhance ingestion activity. When a mixture of macrophages and nonadherent (B and T) cells was treated with 20 μg of lyso-Pc/ml for 30 min, a greatly enhanced Fc-mediated ingestion was observed at about 3 hr after treatment, suggesting that nonadherent cells contributed to activation mechanism of macrophages. The accumulated evidence suggests that treated B cells collaborated with untreated T cells in a stepwise fashion for the exchange of a signaling factor(s) for macrophage activation. When conditioned medium prepared by stepwise cultivation from treated B cells to untreated T cells was used for cultivation of untreated macrophages, a markedly enhanced Fc-mediated ingestion was observed. However, cultivation of macrophages with stepwise conditioned medium of treated T cells and untreated B cells produced no significant enhancement of phagocytic activity. Therefore, we concluded that lyso-Pc-treated B cells initiated the macrophage activation process by releasing and transmitting a signaling factor to T cells, and, in turn, the T cells modified the factor or supplied a new factor capable of the ultimate activation of macrophages for ingestion capacity. This lyso-Pc-induced factor(s) appears to be distinct from the established interleukins 1 and 2.

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.

Characterization of a Nalidixic-Acid-Resistant Mutant of Escherichia coli as a Strict Aerobe

An Escherichia coli mutant, C18, which plates at an efficiency of 5.0 × 10−4 under anaerobic condition, was isolated among spontaneous nalidixic‐acid‐resistant mutants. This strict aerobic mutation was mapped by Pl cotransduction with a gyrA linked transposon Tn10 and found to be at the gyrA gene. A low degree of superhelicity of pBR322 DNA isolated from C18 was demonstrated by agarose gel electrophoresis with various concentrations of ethidium bromide. The superhelical density of pBR322 isolated from C18 was 80% of the value of pBR322 isolated from wild‐type bacteria cultured under aerobic condition, and 50% cultured under anaerobic condition. These results lead us to conclude that a certain mutation of the gyrA gene causes a decrease in DNA superhelicity and prevents anaerobic growth.

Anaerobic expression of the gyrA gene

Abstract E. coli C600Δ lac was infected with a Mu- lac defective phage. Lysogens resistant to ampicillin (25 μg/ml) and nalidixic acid (20 μg/ml) were unable to grow anaerobically and insensitive to nalidixic acid (150 μg/ml), indicating formation of gyrA ::Mu- lac fusions. Mapping of the Mu- lac by P1 phage cotransduction with two different gyrA linked Tn 10 transposon loci suggests that the insertion is located in the gyrA gene. These fusion derivatives form small uncolored colonies on lactose MacConkey agar plates aerobically. When these plates are placed in an anaerobic jar for about 4 h, the colonies turn red indicating that the inserted lac gene is expressed only under anaerobic environment. Thus the gyrA promoter is activated under the anaerobic environment and loss of gyrA activity prevents anaerobic cell growth.

A generalized transducing salmonella phage ES18 can recombine with a serologically unrelated phage Fels 1.

SummarynA long-tailed and generalized transducing Salmonella phage ES18 can recombine with a serologically unrelated phage Fels 1. When ES18 phage stocks grown on Fels 1 lysogens were plated on Fels 1 lysogens resistant to ES18, plaques of a new hybrid phage P181 were found at a frequency of about 10-8. Serological analysis of phage P181 showed that it carries the protein coat of Fels 1 and has no serological cross-reactivity with ES18. Phage P181 expresses the clear plaque morphology similar to that of ES18. ES18 was also found to carry a homology with P22 in at least the c regions. Thus, the c markers (c+, c1, c2 and c3) of P22 can be transferred to ES18. When these ES18 strains were used for isolation of P181 phage, the clear plaque morphology of P181 mimicked that of the P22c marker in the ES18 strain which gave rise to the P181 strain. Salmonella typhimurium strain Q1, lysogenic for P181, is immune to ES18 but not to Fels 1. Furthermore, ES18 lysogens are immune to P181. Therefore it is concluded that the hybrid phage P181 conserves the protein coat of Fels 1 and carries at least the c regions of ES18.nWhen ES18 was grown on Fels 1 lysogens of recombination-deficient (recA) mutants, P181 hybrid particles were found in the ES18 stocks at a frequency of about 10-11. This frequency is about 1000-fold lower than that found in ES18 stocks grown on the wild type hosts lysogenic for Fels 1. Therefore, it is concluded that the bacterial recombination mechanism plays an important role in the formation of P181 by recombination between the prophage Fels 1 and the superinfecting phage ES18.nAlthough phage ES18 belongs to B group phages, it possesses a generalized transducing capability. We have studied a possible origin of ES18.

Photodynamic inactivation of rubella virus enhances recombination with a latent virus of a baby hamster kidney cell line BHK21

Rubella virus is very sensitive to photodynamic action. When tested with 1.2 times10‐5M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m2, inactivation kinetics showed a linear single hit curve with a k value of 1.48 min‐1. Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R‐virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV‐treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV‐RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti‐rubella serum nor by anti‐latent virus serum while type 2 hybrid is neutralized by anti‐latent virus serum.