Anti-inflammatory effects of resveratrol occur via inhibition of lipopolysaccharide-induced NF-κB activation in Caco-2 and SW480 human colon cancer cells.

Resveratrol, a polyphenol abundantly found in grapes and red wine, exhibits beneficial health effects due to its anti-inflammatory properties. In the present study, we evaluated the effect of resveratrol on inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 and SW480 cell lines. In the LPS-treated intestinal cells, resveratrol dose-dependently inhibited the expression of inducible NO synthase (iNOS) mRNA as well as protein expression, resulting in a decreased production of NO. In addition, Toll-like receptor-4 expression was significantly diminished in LPS-stimulated cells after resveratrol pre-treatment. To investigate the mechanisms by which resveratrol reduces NO production and iNOS expression, we examined the activation of inhibitor of κB (IκB) in LPS-stimulated intestinal cells. Results demonstrated that resveratrol inhibited the phosphorylation, as well as the degradation, of the IκB complex. Overall, these results show that resveratrol is able to reduce LPS-induced inflammatory responses by intestinal cells, interfering with the activation of NF-κB-dependent molecular mechanisms.

Organization patterns of the AGFG genes: an evolutionary study.

A number of proteins which are needed for the building of new immunodeficiency virus type 1 virions can only be translated from unspliced virus-derived pre-mRNAs. These unspliced mRNAs are shuttled through the nuclear pores reaching the cytosol when bound to the viral protein Rev. However, as a cellular co-factor Rev requires a Rev-binding protein of the AGFG family (nucleoporin-related Arf-GAP domain and FG repeats-containing proteins). In this article we address the evolution of the AGFGs by analyzing the first section of the coding mRNAs. This contains a "core module" which can be traced from Drosophilae to fish, amphibia, birds, and mammals, including man. In the subfamily of AGFG1 molecules the estimated conservation from Drosophilae to primates is 67% (with limited gaps). In some Drosophilae the core module is preceded by a long stretch of more than 300 coding nucleotides, but this additional module is absent in other Drosophilae and in all AGFG1s of other species. The AGFG2 molecules emerged later in evolution, possibly deriving from a duplication of AGFG1s. AGFG2s, present in mammals only, exhibit an additional module of about 50 coding nucleotides ahead of the core module, which is significantly less conserved (54%, with more remarkable gaps). This additional module does not seem to have homologies with the additional module of Drosophilae nor with the precoding section of AGFG1s. Interestingly, in birds a highly re-edited form of the AGFG1 core module (Gallus gallus, Galliformes) coexists with a typical form of the AGFG1 core module (Taeniopygia guttata, Passeriformes).

An analysis of the human chemokine CXC receptor 4 gene.

In this article we analyze some of the structural characteristics of the coding section and the intron of the human chemokine CXC receptor 4 (a 7-transmembrane receptor) pre-mRNA. In the coding sequence the frequencies of the individual nucleotides do not depart significantly from 0.25, while in the intron the frequencies of the As and Gs are significantly lower and higher, respectively, than expected from a random distribution. Analysis of the pattern of association of nucleotides into triplets or couples shows that some triplets or couples occur with frequencies significantly higher or lower than expected when assuming a random association of nucleotides. In particular, in the intron combinations of the same nucleotide are over-represented. 7-or-more nucleotide repeats occur in both the coding section and the intron with frequencies which exceed the confidence limits for a random distribution. For the coding sequence this is possibly explained by the alternans of relatively similar hydrophobic-coding sections and relatively similar intervening intracellular and extracellular hydrophilic-coding sections. 7-or-more nucleotide repeats in reverse order and in reverse/complemented order occur in the intron, but not in the coding section, with frequencies which significantly exceed a random distribution. The numerous intronic repeats in reverse/complemented order may be of relevance for the secondary structure of the intron and might be one important element of the integrated splicing code.

f-Met-Leu-Phe stimulates nitric oxide production in chick embryo neurons: the role of NF-kB.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) is a major chemotactic factor produced by Escherichia coli and other Gram-negative bacteria. In avian models the fMLP effects and the possible expression of FPRs have been poorly investigated. This report demonstrates that fMLP stimulation in vitro is able to elicit significant cellular responses from 10-day chick embryo nerve cells. Cell treatment with 10(-7) M fMLP at 37 degrees C induces a dramatic increase of nitric oxide (NO) production, after 24, 48, and 72 h, respectively. After 72 h of treatment with 10(-7) M fMLP the maximum nuclear translocation of the NF-kB complex protein p65 is visible, corresponding to the greatest NO production. In this context, 72 h of fMLP stimulation lead to a marked expression of the antiapoptotic protein Bcl-2, involved in cell survival. This suggests that activation of the NF-kB complex plays a protective role in chick neuronal cells treated with fMLP, confirmed by the significant neuronal cells degeneration observed after NF-kB inhibition with the specific inhibitor, TPCK. Overall, these data suggest a possible protective mechanism displayed by neurons against toxic molecules, like NO, released after cell exposure to bacterial products.

CD14 major role during lipopolysaccharide-induced inflammation in chick embryo cardiomyocytes.

CD14 is a surface differentiation antigen that functions as a receptor for bacterial lipopolysaccharide. The cellular signaling events that lead to lipopolysaccharide-induced production of inflammatory mediators are the primary cause of myocardial dysfunction observed in sepsis. Here, we evaluated the role of CD14 in chick embryo cardiomyocytes stimulated with lipopolysaccharide. CD14 expression was detected by confocal laser microscopy observation and by immunoblotting analysis. Moreover, we provided evidence for CD14-dependent functional responses of lipopolysaccharide-stimulated cardiomyocytes in terms of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production. Attenuated TNF-alpha and NO secretion, following anti-CD14 treatment of cardiomyocytes, suggested a role for this receptor in lipopolysaccharide-mediated cell responses. We also evidenced that labeled lipopolysaccharide was internalized and localized next to the Golgi complex, at the level of lysosomes, and in the perinuclear zone. The intracytoplasmatic transport seems to depend on the contractile apparatus, because cell pretreatment with cytochalasin D prevented lipopolysaccharide internalization and reduced both TNF-alpha and NO release. Lipopolysaccharide internalization was dependent on CD14 receptor, since anti-CD14 pre-treatment prevented endotoxin uptake by cardiomyocytes. Results demonstrated: (1) CD14 is expressed on the surface membrane of cardiomyocytes; (2) CD14 is involved in cytoskeletal dependent lipopolysaccharide internalization at specific cytoplasmatic locations; (3) CD14 plays a role in lipopolysaccharide-mediated responses by cardiomyocytes after lipopolysaccharide internalization.

Mutation patterns in the chemokine CXC receptor 4 gene subfamily.

Six representative CXCR4 mRNAs of fish, amphibia, birds, and mammals were selected to study the pattern of conservation/mutation of the individual nt of the coding sequences. According to an arbitrary conservation index ranging from 1 to 6, the indexes of conservation were: 5.04 for the first nt of coding triplets; 5.34 for the second nt of triplets, and 3.75 for the third nt of triplets. The average conservation index of the individual triplets was 4.71. The conservation index of the seven hydrophobic transmembrane domains was 5.60, while the cumulative conservation index of the intracytoplasmic and extracellular domains was 4.63. Separate autocorrelation and power spectral analyses of the series of conservation indexes for the first nt, the second nt and the triplets demonstrated a modest "basic" positive correlation for about the first 20 lags and accordingly some power concentration at the lower frequencies (long periods). Within the triplets, the correlation was studied between the conservation indexes of nt 1 and 2, 1 and 3, and 2 and 3. Correlations of 1 with 3 and 2 with 3 were positive, but in the range of the basic local correlation, whereas the correlation between the first and second nt was significantly higher. This correlation, together with the higher conservation of the second nt as compared to the first (two patterns also found in the formyl peptide receptors), are likely to have been established by selection processes directed towards a functional conservation or a "functional repair."

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis.

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.

Plasma membrane damage sensing and repairing. Role of heterotrimeric G-proteins and the cytoskeleton.

Different toxic agents, derived from bacteria, viruses or cells of the immune system, as well as mechanical forces generated during cell locomotion are able to open pores in the cell plasma membrane. Most of these biological agents operate through specific receptors. We studied the formation and resealing of the "non-specific" plasma membrane pores generated by the mild non-ionic detergent Triton X-100. In HL-60-derived granulocytic cells plasma membrane pore opening after a 1-h treatment with Triton X-100 is documented by entry into the cell of the membrane impermeant dye ethidium bromide. As a consequence of the opening of pores the intracellular K(+) concentration falls dramatically, the cytosolic pH diminishes and the cell membrane is depolarized. Furthermore the cells acquire a polarized morphology, demonstrating the involvement of the actin cytoskeleton. At the Triton concentration used the membrane lesions are progressively repaired and by 8h the impermeability to ethidium bromide is restored and the intracellular K(+) concentration is virtually normal. Following treatments with Triton+Pertussis toxin, Triton+Cytochalasin, or Triton+Pertussis toxin+Cytochalasin the progress of membrane repair is dramatically slowed and is no longer completed by 8h. It is concluded that the membrane damage activates pertussis-sensitive G-proteins which likely act as sensors of the damage, while both G-proteins and the actin cytoskeleton are involved in the membrane repair mechanism.

Expression of UDP-glucuronosyltransferase 1A6 isoform in Caco-2 cells stimulated with lipopolysaccharide.

Glucuronidation is an important metabolic process of detoxification in all vertebrates. The reaction is catalyzed by a multigene family of UDP-glucuronosyltransferases (UGTs) able to convert many xenobiotics and endobiotics (hydrophobic substances) to inactive, water-soluble glucuronides. The UGTs play a protective role, facilitating the elimination of potentially toxic metabolites via urine, bile and feces; therefore, impairment of UGTs may have important toxicological consequences. The regulation of UGTs during bacterial infection or inflammation is not well described. In this study, we investigated the in vitro effect of lipopolysaccharide (LPS) on the expression of the UGT1A6 isoform in human colon carcinoma Caco-2 cells. Results demonstrated a significant down-regulation of UGT1A6 expression, both in terms of mRNA and protein levels, and a reduced UGT activity after LPS exposure of cell cultures, suggesting a role for endotoxins on UGT regulation mechanisms.

Inflammatory responses in embryonal cardiomyocytes exposed to LPS challenge: an in vitro model of deciphering the effects of LPS on the heart.

This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.

Differential conservation of nucleotides and conservation/mutation correlations between nucleotides, with special reference to CXC 1 and 4 and FP receptors involved in immune regulation.

Random mutations of the first nucleotide of a coding triplet alter the hydropathic character of 27 % of the hydrophobic amino acids and of 23 % of the hydrophilic amino acids, while random mutations of the second nucleotide alter the hydropathic character of 82 % of the hydrophobic amino acids and of 47 % of the hydrophilic amino acids. In cases of a change of the hydropathic character, a second random mutation in the previously unmutated first or second nucleotide causes reversion to the original character of an additional 11 % of the originally hydrophobic-coding triplets and an additional 14 % of the originally hydrophilic-coding triplets (on average). Thus, a selection oriented towards the preservation of the hydropathic character of amino acids may be expected to eventually result in a higher conservation of the second nucleotide (as compared to the first). In the case of uncorrected mutations of one of the two first nucleotides, it may be expected that appropriate second mutations in the other unaffected nucleotide will be positively selected. This would result in a positive correlation between the conservation/mutation indexes of the two first nucleotides, as these would be prevailingly either both conserved or both mutated. We examined six groups of coding mRNA sequences: chemokine CXC 1 and 4 and formyl peptide receptors; a group comprising different receptors of the rhodopsin-like superfamily, together with some viral sequences which share significant homologies with these receptors; a group of viral sequences with homologies with the rhodopsin-like receptors; a group of solute carriers. In all the experimental groups the second nucleotide of the triplet was the most conserved and a significant positive correlation existed between conservation/mutation indexes of the two first nucleotides. Similar conservation/mutation patterns could be of more general occurrence in the genome, as a consequence of selection processes.

The HIV-1 Rev binding family of proteins: the dog proteins as a study model.

Various proteins that are required for the building of new complete human immunodeficiency type 1 virions (HIV-1) are coded by unspliced or partly spliced virus-derived mRNAs. HIV-1 has developed special strategies for moving these mRNAs to the cytoplasm to be translated. In the nucleus of the infected cell the virus-derived protein Regulator of expression of viral proteins (Rev) can bind both the viral intron-containing mRNAs and the cellular co-factor HIV-1 Rev binding protein (HRB) and this complex may be shuttled through the nuclear pores. HRB genes have been relatively well conserved during evolution, from Drosophila to humans. However, as a consequence of reading-frame shifts due to nt insertions/deletions, the protein products generated may differ considerably from the prototypal HRB protein, which comprises one Arf-GAP zinc finger domain, several Phenylalanine-Glycine (FG) motifs and four Asparagine-Proline-Phenylalanine (NPF) motifs. This variability is best exemplified by four HRB proteins of the dog, which are discussed here in more detail. The hypothesis is advanced that atypical HRB proteins may not be able to bind Rev and possibly have other, still undetermined, functions. Since the cellular co-factor HRB is essential for viral replication and spread but is not required for cell viability and main bodily functions, it might be an attractive candidate for anti-HIV-1 drug targeting.

Infection with Leishmania infantum Inhibits actinomycin D-induced apoptosis of human monocytic cell line U-937.

Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D-induced apoptosis of the human monocytic cell line U-937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite-free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis-inducing agent. Actinomycin D-induced apoptosis in U-937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U-937 cells via a mechanism involving inhibition of caspase-3 activation. Furthermore, protein kinase C delta (PKC delta) cleavage was increased in actinomycin D-treated U-937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC-mediated pathways by LPG can be implicated in the enhanced survival of the parasites. These results support the claim that promastigotes of L. infantum, as well as its surface molecule, LPG, which is in part released in the culture medium, inhibit macrophage apoptosis, thus allowing intracellular parasite survival and replication.