Effects of herpes simplex virus type 1 infection on immune functions of human neutrophils.

BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.

Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from India

Fatty acids are widely occurring in natural fats and dietary oils and they are known to have antibacterial and antifungal properties. However, little is known on the antibacterial and antifungal properties of the blind-your-eye mangrove (Excoecaria agallocha) and this study for the first time determines the fatty acid composition and the antibacterial and antifungal activities of Fatty Acid Methyl Esters (FAME) of the blind-your-eye mangrove plant found along the coastal areas of south India.

Gorduras naturais e óleos são abundantes em ácidos graxos que apresentam atividade antibacteriana e antifúngica. Entretanto, pouco se sabe sobre a atividade antibacteriana e antifúngica de ésteres metílicos de ácidos graxos de mangue "blind-your-eye" (Excoecaria agallocha). Esse estudo relata, pela primeira vez, a composição em ácidos graxos e a atividade antibacteriana e antifúngica de ésteres metílicos de ácidos graxos (FAME) de mangue "blind-your-eye" encontrado ao longo de áreas costeiras do sul da India.

Effect of alisol B acetate, a plant triterpene, on apoptosis in vascular smooth muscle cells and lymphocytes.

Glucocorticoid-induced apoptosis is a well-recognized physiological regulator of T-cell number and function. Alisol B acetate, a triterpene from Alisma Plantago-aquatica, has a glucocorticoid-like structure, and may have a similar function like glucocorticoid-induced apoptosis in both vascular smooth muscle cell line (A7r5) and human acute lymphoblastic leukemia cell line (CEM cells). For exploring its mechanism, mitochondria membrane potential and apoptosis-related gene expression were discussed. Alisol B (10(-6)-10(-4) M) inhibited serum-stimulated DNA synthesis in a concentration-dependent manner (IC50) = 4.0 +/- 0.8 x 10(-6) M in A7r5 and 2.1 +/- 1.2 x 10(-6) M in CEM cells). The cell viability was reduced at 10(-4) M of alisol B. Similar results were seen in dexamethasone treatment (a synthetic glucocorticoid, 10(-6) M, 48 h). Apoptosis was induced after the cells were exposed to 10(-5)-10(-4) M alisol B or 10(-6) M dexamethasone for 48 h. The mitochondrial membrane potential (delta psi(m)) was significantly reduced after the alisol B treatment, indicating that the mitochondria might play a role in the alisol B induced cell apoptosis. Alisol B (10(-5)-10(-4) M) increased the levels of c-myc and bax mRNA and proteins, but not on the anti-apoptotic proto-oncogene, bcl-2, in A7r5 and CEM cells. In contrast, dexamethasone (10(-6) M) treatment only caused significant increase in c-myc mRNA levels. These results suggest that the increased ratio of Bax/Bcl-2 and the decreased mitochondrial membrane potential might be involved in the mechanisms of alisol B-induced cell apoptosis.

Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA.

1. The mechanisms involved in the apoptotic effect of saikosaponin-d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3 x 10(-7) M). 2. Saikosaponin-d (10(-8) to 10(-5) M) inhibited the serum-stimulated [(3)H]-thymidine incorporation in a concentration-dependent manner. Dexamethasone also inhibited serum-stimulated [(3)H]-thymidine incorporation. 3. Cell viability was unaffected by saikosaponin-d until 10(-5) - 10(-4) M. Dexamethasone significantly reduced the number of viable cells. 4. Following saikosaponin-d (10(-5) - 10(-4) M) treatment, flow cytometry analysis of propidium iodide-stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin-d (10(-5) - 10(-4) M) induced apoptosis as well as necrosis. 5. The apoptotic effect of saikosaponin-d (3 x 10(-6) - 10(-4) M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin-d (3 x 10(-6) - 10(-5) M) was unaffected by the presence of Z-VAD-FMK, indicating that saikosaponin-d-induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z-VAD-FMK. 6. Levels of c-myc, p53, and bcl-2 mRNA were analysed by the reverse transcription-polymerase chain reaction. Levels of c-myc and p53 mRNA were significantly increased, while the level of bcl-2 mRNA was decreased, by saikosaponin-d (10(-5) M) treatment. Dexamethasone did not significantly change the expression of these genes. 7. It is suggested that the apoptotic effect of saikosaponin-d may be partly mediated by increases in c-myc and p53 mRNA levels accompanied by a decrease in bcl-2 mRNA level.

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.

Insulin-degrading enzyme in a human colon adenocarcinoma cell line (Caco-2).

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.

Biosynthesis of the immunosuppressant immunomycin: the enzymology of pipecolate incorporation.

Immunomycin, an immunosuppressant closely related to FK 506, contains a pipecolate residue in amide linkage with an acyl group in its polyketide backbone. An enzyme activating L-pipecolic acid has been isolated from Streptomyces hygroscopicus var. ascomyceticus, which produces immunomycin. Purification results in a monomer of 170 kDa exhibiting N-terminal heterogeneity, apparently arising from proteolysis of a single species. It is a dimer under native conditions. The reaction appears to use an aminoacyl adenylate as an intermediate in the activating reaction, as do most activating enzymes involved in nonribosomal peptide synthesis. A range of pipecolate and proline analogues act as substrates in the pyrophosphate-ATP exchange resulting from the adenylation reaction. Several analogues are inhibitors of the subsequent thioesterification of the enzyme. Antibody raised to the purified enzyme was used to follow antigen during the course of fermentation. Maximal levels of antigen are found when synthesis of immunomycin is maximal. Ten of twelve immunomycin nonproducing mutants lack detectable pipecolate-activating enzyme in Western blots. From the enzymatic characteristics, substrate specificity, and immunological properties, we propose that we have isolated the enzyme responsible for activating pipecolic acid for immunomycin biosynthesis.

Interactions of polymerized phospholipid vesicles with cells. Uptake, processing and toxicity in macrophages.

We have studied the uptake of photopolymerized multilamellar vesicles composed of bis(1,2(methacryloyloxy)dodecanoyl)-L-alpha-phosphatidylchol ine (DPL) by mouse peritoneal macrophages in vitro. Vesicles composed of polymerized DPL are taken up more rapidly and extensively than vesicles composed of conventional phosphatidylcholine. The uptake of radioactive DPL vesicles was not blocked by incubation with unlabelled phosphatidylcholine vesicles in either the fluid or gel state. Likewise, fluid-phase negatively charged vesicles failed to block uptake of DPL vesicles, whereas solid-phase negatively charged vesicles did have a blocking effect. A radioactive lipophilic marker (dipalmitoylphosphatidyl[N-methyl-3H]choline) incorporated into DPL vesicles was metabolized at essentially the same rate whether the vesicles were polymerized or not. Nonpolymerized DPL vesicles were quite toxic to macrophages, whereas polymerized DPL vesicles or vesicles composed of conventional phosphatidylcholines were not toxic.

Interactions of conventional or photopolymerized liposomes with platelets in vitro.

We have examined the effects of liposomes on in vitro platelet aggregation. Liposomes were prepared from various conventional lipids and from a novel photopolymerizable phosphatidylcholine derivative (DPL, bis[1,2-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine). None of the liposome preparations studied caused marked platelet aggregation in either plasma or buffer solution. However, positively charged vesicles impaired the ability of platelets in plasma to aggregate in response to ADP, whereas negatively charged vesicles impaired the ability of platelets in buffer to aggregate in response to thrombin. DPL vesicles had only modest effects on platelets in plasma or buffer.

Interactions of liposomes with the reticuloendothelial system. II: Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages.

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charge vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipids, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10(2)-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.