Transmission enhancement through a trench-surrounded nano metallic slit by bump reflectors.

We propose using a pair of bumps bordering the conventional trench-surrounded metal nano slit in order to confine the surface waves and further enhance the slit transmission. The bump height of 1.mum is larger than the depth of penetration on air side of the surface waves. The reflectivity of such bumps is larger than 95%. A very large slit transmission, which is 50% of the energy of the incident beam impinging on the entire size 13.mum of the trench-surrounded slit structure, is obtained through the metallic slit of 50nm width and 400nm depth. The bumps enhance the transmission by 1.75 fold.

Distribution of human chemokine (C-X3-C) receptor 1 (CX3CR1) gene polymorphisms and haplotypes of the CC chemokine receptor 5 (CCR5) promoter in Chinese people, and the effects of CCR5 haplotypes on CCR5 expression.

Two chemokine (C-X3-C) receptor 1 (CX3CR1) gene polymorphisms, V249I and T280M, and 10 CC chemokine receptor 5 (CCR5) promoter haplotypes, P1-P10, have recently been reported to influence the progression of acquired immune-deficiency syndrome (AIDS). As these studies were performed mainly with Caucasian and African-American subjects, we determined the distribution of these alleles in Chinese people for the purpose of predicting possible clinical responses to the human immunodeficiency virus type 1 (HIV) epidemics in countries with significant Chinese populations, as well as to establish their effects on the expression of surface CCR5. Ninety-six HIV-negative Chinese individuals in Taiwan were subjected to genotyping, and we thus determined that the allelic frequencies of CX3CR1V249I and T280M changes were 2.6% and 2.1%, respectively, which were lower than found in Caucasians (25.5% and 14.0%, respectively). Unlike the previous reports, we only detected CCR5P1 and P4 haplotypes in Taiwanese people, and the P1/P1, P1/P4 and P4/P4 genotype frequencies were 21.0%, 41.1% and 37.9%, respectively. The sequencing data confirmed the results of previous studies, showing that CCR5P1 exhibited a complete linkage disequilibrium with a polymorphic allele 59029A present in the CCR5 promoter. Furthermore, fluorescence-activated cell sorter analysis revealed that, in the absence of the CCR2-64I mutation, individuals carrying CCR5P1 tended to express more surface CCR5 on monocytes and CD4+ cells. Therefore, this study not only reports the frequencies for the CX3CR1 and CCR5 promoter haplotypes in a Chinese population living in Taiwan, but also identifies a statistical link between the P1/P1 haplotype and the elevated CCR5 expression levels in the study group.

Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)

In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties.

Curcumin is a naturally occurring, dietary polyphenolic phytochemical that is under preclinical trial evaluation for cancer preventive drug development and whose working pharmacological actions include anti-inflammation. With respect to inflammation, in vitro, it inhibits the activation of free radical-activated transcription factors, such as nuclear factor kappaB (NFkappaB) and AP-1, and reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha), interleukin-1beta (IL-1beta), and interleukin-8. Inducible nitric oxide synthase (iNOS) is an inflammation-induced enzyme that catalyzes the production of nitric oxide (NO), a molecule that may lead to carcinogenesis. Here, we report that in ex vivo cultured BALB/c mouse peritoneal macrophages, 1-20 microM of curcumin reduced the production of iNOS mRNA in a concentration-dependent manner. Furthermore, we demonstrated that, in vivo, two oral treatments of 0.5 mL of a 10-microM solution of curcumin (92 ng/g of body weight) reduced iNOS mRNA expression in the livers of lipopolysaccharide(LPS)-injected mice by 50-70%. Although many hold that curcumin needs to be given at dosages that are unattainable through diet to produce an in vivo effect, we were able to obtain potency at nanomoles per gram of body weight. This efficacy is associated with two modifications in our preparation and feeding regimen: 1) an aqueous solution of curcumin was prepared by initially dissolving the compound in 0.5 N NaOH and then immediately diluting it in PBS; and 2) mice were fed curcumin at dusk after fasting. Inhibition was not observed in mice that were fed ad lib., suggesting that food intake may interfere with the absorption of curcumin.

Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea.

Chronic inflammation has been implicated as the underlying factor in the pathogenesis of many disorders. In the past decade, inflammation-related endogenous production of reactive nitrogen species, similar to oxygen free radicals, has also been suggested as a risk factor for cancer, in addition to the well-studied exogenous nitroso compounds. Epidemiological, in vitro, and animal model studies have implicated green tea to be protective against nitroso compound-induced and inflammation-related cancer. Therefore, we investigated the effect of epigallocatechin-3-gallate (EGCG), one of the known biologically active catechins contained in green tea, on the production of nitric oxide (NO.). We have shown previously that EGCG reduces NO. production as measured by nitrite accumulation in the culture medium. Expanding on this finding, in this report we show that EGCG may do so by two mechanisms: reduction of inducible nitric oxide synthase (iNOS) gene expression and inhibition of enzyme activity. Addition of 1-10 microM EGCG to lipopolysaccharide- and interferon-gamma-activated mouse peritoneal cells reduced iNOS mRNA expression concentration dependently, to 82-14%, as measured by relative reverse transcription-polymerase chain reaction. Addition of 50-750 microM EGCG, in a concentration-dependent manner, inhibited the enzyme activity of iNOS, to 85-14%, and neuronal nitric oxide synthase (nNOS), to 93-56%, as measured by citrulline formation. EGCG competitively inhibited binding of arginine and tetrahydrobiopterin, and the gallate structure is important for this action.

Effects of three dietary phytochemicals from tea, rosemary and turmeric on inflammation-induced nitrite production.

In chronic inflammation, cytokines induce the production of nitric oxide (NO.) that is converted to DNA damaging and carcinogenic peroxynitrite and nitrite. The compounds epigallocatechin gallate (EGCG), carnosol, and curcumin are non-vitamin phytochemicals contained in commonly consumed dietary plants. They are known to be anti-inflammatory and cancer preventive. Therefore, we studied their effect on the generation of peroxynitrite radicals and nitrite. They inhibited lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) induced nitrite production by mouse peritoneal cells by more than 50% at 2.5-10 microM. Cell viability assays verified that the inhibition was not due to general cellular toxicity.

Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon.

MAP30 is an anti-HIV plant protein that we have identified and purified to homogeneity from bitter melon (Momordica charantia). It is capable of acting against multiple stages of the viral life cycle, on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase and cell-free ribosome-inactivation activities. We have cloned and expressed the MAP30 gene. The objective of this study is to characterize recombinant MAP30 (re-MAP30) and to determine its anti-HIV, anti-tumor and other activities. We report here that re-MAP30 inhibits HIV-1 and certain human tumors to the same extent as its native counterpart, natural MAP30 (nMAP30). The anti-HIV activity was measured by quantitative focal syncytium formation on CEM-ss cell monolayers, viral core protein p24 expression and viral-associated reverse transcriptase activity in HIV-1-infected H9 cells. The anti-tumor activity was measured by metabolic labeling of protein synthesis in tumor cells. In the dose range of the assay, re-MAP30 exhibits little toxicity to the uninfected viral target cells and other normal human cells. Identical to nMAP30, re-MAP30 is also active in topological inactivation of viral DNA, inhibition of viral DNA integration and cell-free ribosome inactivation. The cloning and expression of the gene encoding biologically active re-MAP30 provides an abundant source of homogeneous material for clinical investigations, as well as structure-function studies of this novel antiviral and anti-tumor agent.

Human immunodeficiency virus type 1 (HIV-1) inhibition, DNA-binding, RNA-binding, and ribosome inactivation activities in the N-terminal segments of the plant anti-HIV protein GAP31.

GAP31 (gelonium anti-HIV protein of 31 kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity. The ability of GAP31 to interrupt both DNA and RNA functions may be related to its multiple antiviral actions. To define the roles of these activities in the anti-HIV action of GAP31, a series of peptides corresponding to the N-terminal segment of GAP31 were synthesized and assayed for the aforementioned activities of the parent molecule. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) designated as K10-K42 is the shortest peptide necessary and sufficient for HIV-1 inhibition, DNA and RNA binding, and ribosome inactivation. The peptides were 2-5 orders of magnitude less active than GAP31. Truncation of 19 aa from the C terminus of K10-K42 resulted in the loss of all of these activities. On the other hand, deletion of N-terminal residues to give E23-K42 did not alter ribosome-inactivation activity but eliminated the other activities. These findings permit identification of a 7-aa sequence, KGATYIT, at the N terminus of K10-K42 that is critical for DNA binding and RNA binding, whereas a 9-aa sequence, SHGIPSLRK, at the C terminus is important to ribosome inactivation. Both regions contribute to anti-HIV activity. Histidine at position 35 is critical for all of these activities. The disparity of sequence requirements for inhibition of HIV infection and replication and for ribosome-inactivation activity suggests that the anti-HIV activity of most ribosome-inactivating proteins may not be the result of N-glycosidase activity alone. Mapping the minimal domain of GAP31 offers insights into the rational design of molecular mimetics of anti-HIV drugs.

Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms.

GAP 31, DAP 32 and DAP 30 comprise a new class of plant proteins with potent anti-HIV activity and insignificant cytotoxicity. We report here the identification and characterization of a new DNA enzyme activity in these three proteins. They irreversibly relax and decatenate supercoiled DNA, as well as catalyze double-stranded breakage to form linear DNA. The relaxed molecules are topologically inactive and no longer serve as substrates for DNA gyrase to form supercoils, phenomena similar to those of cellular topoisomerases in the presence of topoisomerase poisons. The ability of these anti-HIV agents to interrupt essential topological interconversions of DNA may provide a novel mechanism for their antiviral and antitumor actions. The presence of this new DNA topological enzyme activity in these plant proteins also suggests that their anti-HIV activity may not be merely a consequence of ribosome inactivation previously recognized.

A new class of anti-HIV agents: GAP31, DAPs 30 and 32.

Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds (Gelonium multiflorum) and carnation leaves (Dianthus caryophyllus). These proteins, GAP 31 (Gelonium Anti-HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti-HIV proteins, 30 and 32 kDa), inhibit HIV-1 infection and replication in a dose-dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 10(4), suggesting that they may be clinically important agents in the treatment of AIDS. The N-terminal amino acid sequences of these proteins show little homology to those of previously described anti-HIV proteins. The structure-function features of these HIV inhibitors, based on the 40-60 amino acid residues of N-terminal sequences, are examined.

TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells.

An anti-human immunodeficiency virus (anti-HIV) protein capable of inhibiting HIV-1 infection and replication has been isolated and purified to homogeneity from Trichosanthes kirilowii. This protein, TAP 29 (Trichosanthes anti-HIV protein, 29 kDa), is distinct from trichosanthin [also known as GLQ 223 (26 kDa)] in size, N-terminal amino acid sequence, and cytotoxicity. In addition to three conservative substitutions--namely, Arg-29 to Lys, Ile-37 to Val, and Pro-42 to Ser--a total difference of residues 12-16 was found. TAP 29 yielded -Lys-Lys-Lys-Val-Tyr-, whereas trichosanthin has -Ser-Ser-Tyr-Gly-Val-. Although the two proteins exhibit similar anti-HIV activity, as measured by syncytium formation, p24 expression, and HIV reverse transcriptase activity, they differ significantly in cytotoxicity, as measured by their effects on cellular DNA and protein syntheses. At the dose level of the bioassays, 0.34-340 nM, trichosanthin demonstrates a dose-dependent toxic effect on host cells. TAP 29 displays no toxic effect, even at 100 X ID50, whereas trichosanthin demonstrates 38% and 44% inhibition on cellular DNA and protein synthesis, respectively. These results indicate that the therapeutic index of TAP 29 is at least two orders of magnitude higher than that of trichosanthin. Thus TAP 29 may offer a broader safe dose range in the treatment of AIDS.

MAP 30: a new inhibitor of HIV-1 infection and replication.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (ii) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

Formation of mutagens in boiled pork extract.

When boiled pork extract was heated under reflux at 102 degrees C for 4 hr mutagens, which were detected using Salmonella typhimurium strains TA98 and TA1538, were formed. The level of mutagenicity was dependent on the concentration of pork in the extract, the duration of boiling and on pH; the optimum pH for mutagen formation was found to be 9 to 11. Thin-layer chromatographic analysis showed that the mutagens formed in boiled pork extract were chromatographically distinguishable from benzo[a]pyrene and from the primary mutagenic pyrolysis products of tryptophan (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and of glutamic acid (2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole).