Arl4c expression in colorectal and lung cancers promotes tumorigenesis and may represent a novel therapeutic target.

We recently demonstrated that expression of ADP-ribosylation factor (ARF)-like 4c (Arl4c) induced by a combination of Wnt/ß-catenin and epidermal growth factor/Ras signaling in normal epithelial cells grown in three-dimensional culture promotes cellular migration and proliferation, resulting in formation of tube-like structures, suggesting the involvement of Arl4c in epithelial morphogenesis. It is conceivable that there could be a common mechanism between epithelial morphogenesis and carcinogenesis. Therefore the current study was conducted to investigate whether Arl4c might be involved in tumorigenesis. Immunohistochemical analyses of tissue specimens obtained from colorectal and lung cancer patients revealed that Arl4c was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor lesions. Inhibition of Wnt/ß-catenin or Ras/mitogen-activated protein kinase signaling reduced Arl4c mRNA levels in HCT116 colorectal cancer cells and A549 lung cancer cells. Knockdown of Arl4c inhibited Rac activity and also prevented nuclear localization of yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) in these cancer cells. Arl4c-depleted cancer cells consistently showed decreased migration, invasion and proliferation capabilities both in vitro and in vivo. Furthermore, direct injection of Arl4c small interfering RNA (siRNA) into HCT116 cell-derived tumors (in vivo treatment with siRNA) inhibited tumor growth in immunodeficient mice. These results suggest that Arl4c is involved in tumorigenesis and might represent a novel therapeutic target for suppressing proliferation and invasion of colorectal and lung cancer cells.

[Simultaneous resection of pulmomary metastasis of colon cancer and primary lung cancer].

A 70-year-old woman was admitted with a complaint of weight loss and an abnormal shadow on the chest X-ray. On palpation, the unmobilized mass, measuring 5 cm, was located in the left lower abdomen. The barium-enema examination showed the filling defect of the sigmoid colon. Chest computed tomography (CT) showed a tumor, measuring 20 x 20 mm, located in the right upper lobe (S3) and a nodule, measuring 3 mm, located in the right lower lobe (S8). At first, we performed sigmoidectomy (D 3) for the colon cancer. Next, performed right upper lobectomy and a partial resection of the right lower lobe. Histopathologically, the one is a primary lung cancer, the other is a metastatic lung cancer. With an increase in colorectal and lung cancer, similar cases as ours seem to increase in number. When we treat multiple lung nodules with malignancy of other organs, we should consider 3 types of cases, 1) only primary, 2) primary and metastatic, 3) only metastatic.

Induction of terminal differentiation and apoptosis in human colonic carcinoma cells by brefeldin A, a drug affecting ganglioside biosynthesis.

An appreciable increase in G(M3) with a concomitant decrease in some neolacto-series gangliosides was observed during differentiation of human colonic carcinoma HCT 116 cells induced by a differentiating agent. When the cells were treated with brefeldin A (BFA), a striking increase in de novo biosynthesis of G(M3) and a decrease in biosynthesis of neolactoseries gangliosides were observed after 6 h. Clear morphological changes to differentiated epithelial cells and an arrest of cells in the G0/G1 phase of the cell cycle were observed after 1 day of treatment. Then the cells were led to apoptosis. This activity was not affected by forskolin, which antagonizes the effects of BFA on protein transport and the Golgi apparatus. These results suggest that the differentiation-inducing activity of BFA might be due to its modulatory effect on ganglioside biosynthesis, and that a specific change in ganglioside pattern is an essential prerequisite for induction of differentiation, providing a novel target for differentiation therapy of cancer.

Activated mast cells release extracellular type platelet-activating factor acetylhydrolase that contributes to autocrine inactivation of platelet-activating factor.

IgE-dependent and -independent activation of mouse bone marrow-derived mast cells (BMMC) elicited rapid and transient production of platelet-activating factor (PAF), which reached a maximal level by 2-5 min and was then degraded rapidly, returning to base-line levels by 10-20 min. Inactivation of PAF was preceded by the release of PAF acetylhydrolase (PAF-AH) activity, which reached a plateau by 3-5 min and paralleled the release of beta-hexosaminidase, a marker of mast cell exocytosis. Immunochemical and molecular biological studies revealed that the PAF-AH released from activated mast cells was identical to the plasma-type isoform. In support of the autocrine action of exocytosed PAF-AH, adding exogenous recombinant plasma-type PAF-AH markedly reduced PAF accumulation in activated BMMC. Furthermore, culture of BMMC with a combination of c-kit ligand, interleukin-1beta and interleukin-10 for > 24 h led to an increase in plasma-type PAF-AH expression, accompanied by a reduction in stimulus-initiated PAF production. Collectively, these results suggest that plasma-type PAF-AH released from activated mast cells sequesters proinflammatory PAF produced by these cells, thereby revealing an intriguing anti-inflammatory aspect of mast cells.

Separation of 18 6-aminoquinolyl-carbamyl-amino acids by ion-pair chromatography.

Eighteen 6-aminoquinolyl-carbamyl (AQC)-amino acids were separated by ion-pair chromatography, using tetrabutylammonium (TBA) as a counterion. Optimum separation was obtained on a C8 reverse-phase column using gradient elution with two mobile phases, A (5 mM TBA, 75 mM ammonium acetate, pH 7.5) and B (80% acetonitrile). The AQC-amino acids were detected by fluorescence with excitation at 250 nm and emission at 395 nm, and the analysis time was 65 min. The response factors of individual AQC-amino acids to AQC-phenylalanine ranged from 0.42 to 1.08 (except for tryptophan at 0.01), with an average of 0.8. Detection limits by fluorescence ranged from 11.8 fmol (threonine) to 51.7 fmol (methionine), except for tryptophan (1.8 pmol).

A distribution study of 11C platelet-activating factor (PAF) analogs in normal and tumor-bearing mice.

As a preliminary study to image platelet-activating factor (PAF) receptors in vivo, comparative study of biodistribution between 1-O-hexadecy1-2-O-N, N-dimethylcarbamoyl-sn-glycero-3-phosphocholine [choline-methyl-11C](L-[11C]dimethylcarbamoyl-PAF) and nonspecific PAF analog, 3-O-hexadecyl-2-O-N,N-dimethylcarbamoyl-sn-glycero-1-phosphocholine [choline-methyl-11C](D-[11C]-dimethylcarbamoyl-PAF) was carried out in both normal and tumor-bearing mice. Higher accumulation of L-[11C]dimethylcarbamoyl-PAF than D-[11C]dimethylcarbamoyl-PAF was observed in normal mice spleen. The co-administration of PAF antagonists dose-dependently reduced the radioactivity level of the L-isomer only in the spleen. In mice bearing Ehrlich tumors and Sarcoma 180, more L-than the D-[11C]-isomer was accumulated in the tumor and spleen. We found that specific accumulation sites for L-[11C]dimethylcarbamoyl-PAF exist in the spleen and tumors than in other tissues. Moreover, the comparison of accumulation between L- and D-[11C] dimethylcarbamoyl-PAF would be a useful procedure for estimation of PAF receptors in vivo.

Clear differences in ceramide metabolism between glycosphingolipids and sphingomyelin in a human promyelocytic leukemia cell line HL-60 stimulated by a differentiation inducer.

Although the ceramide components of both glycosphingolipids (GSLs) and sphingomyelin (SM) in HL-60 cells were identical, the molecular species of the ceramides preferentially used in biosynthesis were quite different in GSLs and SM. When HL-60 cells were stimulated to differentiate into macrophage-like cells by phorbol ester after their sphingolipids had been metabolically labeled with L-[3-14C]serine to saturation point, marked changes in the radioactivities of the ceramide residues were observed in GSLs, showing the activation of a biosynthetic pathway of ganglioside GM3. No significant changes were, however, observed in the ceramide residues of SM. These results indicate that it is necessary to consider the overall metabolism of ceramides, including their origin, when investigating the functions of ceramides in signal transduction systems.

Selective inhibition of human TNF-alpha action by flecainide acetate, an antiarrhythmic drug.

It is now generally accepted that human tumor necrosis factor-alpha (hTNF-alpha) affects not only tumor cells but also normal cells, providing critical tissue damage. hTNF-alpha also enhanced the response of polymorphonuclear neutrophils (PMN) by its priming action and resulted in the increased generation of active oxygen which in turn may be responsible for the tissue injury. Seeking a conventional drug to attenuate the cytolytic activity of tumor necrosis factor (TNF-alpha) and thereby prevent excessive tissue injury, we focused on the cytolytic action of hTNF-alpha against L929 cells, which are sensitive to TNF-alpha, and found that flecainide acetate [N-(2-piperidylmethyl) 1,5-bis-(2,2,2-trifluoroethoxy) benzamide acetate] inhibited specifically the cytolytic action of hTNF-alpha against L929 cells. Flecainide acetate also specifically inhibited the priming action of hTNF-alpha which enhance the formylmethionyl-leucyl-phenylalanine (FMLP)-induced receptor-mediated superoxide (O.2-) generation of human peripheral polymorphonuclear neutrophils (hPMN). The ID50 values for hTNF-alpha induced cytotoxicity in L929 cells and hTNF-alpha primed FMLP-induced O.2- generation of hPMN were 30 and 50-60 microM, respectively. However, the drug does not inhibit the FMLP- or phorbol myristate acetate (PMA)-induced O.2- generation of nonprimed hPMN and has a weak cytotoxic effect on L929 cells. From these results, it is concluded that flecainide acetate suppressed specifically the action of hTNF-alpha.

Purification and characterization of platelet-activating factor acetylhydrolase from peritoneal fluid obtained from guinea pigs after endotoxin shock.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetyl-hydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.

Platelet-activating factor (PAF) and the development of chronic subdural haematoma.

Platelet activating factor (PAF) content and PAF-acetylhydrolase (PAFAH) activity were measured in the plasma and haematoma of 34 chronic subdural haematoma (CSH) patients. The plasma PAF level in patients with CSH was higher than that in healthy controls. Although there was no correlation between the plasma PAF levels and the interval between the onset of symptoms and the day of sampling, namely, the interval after bleeding, the haematoma PAF level gradually decreased according to the interval after the onset of symptoms. There was no difference between plasma PAFAH activity in patients with CSH and that in healthy controls, and haematoma enzyme activity gradually increased correlated with the interval between the onset of symptoms and surgery. In addition, the localization of PAF in haematoma capsules was histochemically determined. PAF was solely localized to the peri-sinusoidal vessels in the outer membrane of haematoma capsules. Based on these biochemical and histochemical studies, we speculated that PAF may play a role in the development of chronic subdural haematomas.

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.

Biological response of guinea pig peritoneal macrophages to platelet-activating factor.

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9 x 10(4)/cell) with a dissociation constant of 2.3 x 10(-10) M. When treated with 10(-9)-10(-5) M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10(-5) M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated by N-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 microM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H2O2 release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-beta-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10(-5) M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response).

Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

A novel bioaction of PAF: induction of microbicidal activity in guinea pig bone marrow cells.

When guinea pig bone marrow cells were incubated in the presence of 10(-8) to 10(-6) M platelet activating factor (PAF) for 24 to 72 hr, microbicidal activity against Candida parapsilosis of cells was augmented. This augmentation was inhibited by PAF-specific antagonists, CV6209 or FR900452. PAF-specific binding sites with a high affinity were found on guinea pig bone marrow cells. Carrageenan or 2-chloroadenosine, reagents known to be preferentially cytotoxic to macrophages, abolished the microbicidal activity of PAF-treated bone marrow cells. Macrophages prepared from the peritoneal cavity, however, acquired no appreciable microbicidal action by treatment with PAF. These observations suggest that PAF may affect a class of guinea pig bone marrow cells through binding to receptors specific to PAF, resulting in activation and/or induction of differentiation of monocyte-macrophage lineage cells.

Lack of protein-mediated alpha-tocopherol transfer between membranes in the cytoplasm of ascites hepatomas.

Transfer-stimulating activity for alpha-tocopherol and the concentration of alpha-tocopherol and peroxidized lipids in rat ascites hepatoma cells were compared with those from normal and regenerating liver. The ability of supernatants from ascites hepatomas (AH-13, AH-60C, AH-109A) to enhance the transfer of alpha-tocopherol was much lower than that from normal livers. The alpha-tocopherol per mg protein of supernatant from ascites hepatomas was lower than that from normal liver. Regenerating liver showed almost the same values as normal liver in activity to stimulate the transfer of alpha-tocopherol and alpha-tocopherol content of the supernatant. By gel filtration, about 60% of alpha-tocopherol in the supernatant of normal liver was detected in the fractions containing the 30 K protein, which stimulates transfer of alpha-tocopherol between membranes, whereas no significant amount of alpha-tocopherol was detected in 30 K protein fractions of AH-60C supernatant. Little stimulating activity for alpha-tocopherol transfer was detected in AH-60C, AH-109A and AH-13. All ascites hepatomas tested contained less arachidonic acid and docosahexaenoic acid than normal and regenerating liver. An absorption peak with maximum intensity at 233 nm, which is due to conjugated dienes, was observed in UV-absorption spectra of ascites hepatoma total lipids, indicating that peroxidized lipids accumulate in these cells. With normal and regenerating liver, no significant peak due to conjugated dienes was detected.

Antitumor activity of synthetic alkylphospholipids with or without PAF activity.

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycero-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe with sn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by the sn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by "nonspecific" mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar "nonspecific" mechanisms.

Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2.

The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.

Activation of mouse peritoneal macrophages by synthetic glyceroglycolipid liposomes.

Liposomes composed of chemically synthesized glyceroglycolipids, such as 1,2-dipalmityl-[beta-cellobiosyl-(1'----3)]- glycerol (Cel-DAG), 1,2-dipalmityl-[beta-lactosyl-(1'----3)]-glycerol, or 1,2-dipalmityl-[beta-maltosyl-(1'----3)]-glycerol, were found to enhance protective immunity against transplantable tumor cells (sarcoma 180) in ICR mice. Peritoneal exudate cells prepared from mice treated in vivo with Cel-DAG showed cytostatic activity in vitro against the mouse leukemia cell line, EL-4. Adherent cells separated from this preparation showed similar activity. Peritoneal cells from polypeptone-injected mice acquired appreciable cytostatic activity when incubated in vitro in the presence of glyceroglycolipid liposomes. The adherent cell fraction alone showed rather weak cytostatic activity when pretreated with the glyceroglycolipids, and full activity was restored by supplementing with the nonadherent cell fraction. The ability of glycolipids to induce tumoricidal effects was affected by cholesterol content: with increasing cholesterol content, the activities decreased. Cholesterol-free glycolipid liposomes were taken more efficiently by macrophages than cholesterol-containing liposomes. Cholesterol modifies the surface property of glyceroglycolipid liposomes. Activation of macrophages is responsible for enhancement of protective immunity against tumor cells by injection of these glycolipids in vivo.

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists.

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes.

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.