Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Iron Overload Causes Alterations of E-Cadherin in the Liver.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Inhibitory effects of Bcl-2 on mitochondrial respiration.

In contrast to the well-established anti-apoptotic effect of Bcl-2 protein, we have recently demonstrated that Bcl-2 overexpression by vaccinia virus causes apoptosis in BSC-40 cells, while it prevents apoptosis in HeLa G cells. Given the key role of mitochondria in the process of apoptosis, we focused on effects of Bcl-2 expression on mitochondrial energetics of these two cell lines. In this study we present data indicating that BSC-40 cells derive their ATP mainly from oxidative phosphorylation whereas HeLa G cells from glycolysis. More importantly, we show that in both cell lines, Bcl-2 inhibits mitochondrial respiration and causes a decrease of the ATP/ADP ratio. However, it appears that BSC-40 cells cannot sustain this decrease and die, while HeLa G cells survive, being adapted to the low ratio of ATP/ADP maintained by glycolysis. Based on this observation, we propose that the outcome of Bcl-2 expression is determined by the type of cellular ATP synthesis, namely that Bcl-2 causes apoptosis in cells relying on oxidative phosphorylation.

Protooncogene Bcl-2 induces apoptosis in several cell lines.

Using a recombinant vaccinia virus expressing protooncogene Bcl-2, we demonstrate opposite effects of the expressed Bcl-2 in two cell lines: apoptosis induction in BSC-40 cells and apoptosis prevention in HeLa G cells. The apparent molecular weight of the expressed Bcl-2, its amounts and its effects on the mitochondrial membrane potential are comparable in both cell lines, suggesting that the consequences of Bcl-2 expression depend on the cellular environment. To further support these findings we demonstrate the pro-apoptotic effect of the expressed Bcl-2 in several other cell lines.

Detection of galectin-3 in tear fluid at disease states and immunohistochemical and lectin histochemical analysis in human corneal and conjunctival epithelium.

BACKGROUND/AIM: Components of the tear fluid contribute to the biochemical defence system of the eye. To reveal whether the immune mediator and lipopolysaccharide binding galectin-3 is present in tears, tear samples were collected from eyes in healthy and pathological states. Investigation of expression of galectin-3 and galectin-3 reactive glycoligands in normal human conjunctival and corneal epithelia was also initiated as a step to understand the role of galectin-3 in ocular surface pathology. METHODS: Immunoblot analysis using either a rabbit polyclonal or a mouse monoclonal antibody against galectin-3 was employed to detect galectin-3 in tear fluid. Galectin-3 expression in tissue specimens was detected by immunocytochemistry employing A1D6 mouse monoclonal antibody, and galectin-3 reactive glycoligands were visualised by lectin histochemistry using labelled galectin-3. RESULTS: Galectin-3 was found only in tears from patients with ocular surface disorders. It was expressed in normal corneal and conjunctival epithelia but not in lacrimal glands. Inflammatory leucocytes and goblet cells found in galectin-3 containing tear fluid also expressed galectin-3. Galectin-3 binding sites were detected on the surface of conjunctival and corneal epithelial cells co-localising with desmoglein. CONCLUSIONS: This study revealed expression of galectin-3 in tear fluid obtained from patients with eye diseases. The role of this endogenous lectin (produced by inflammatory as well as epithelial cells) in antimicrobial action and inflammation modulation could be expected.

Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme.

Induction of apoptosis in mammalian cells by double-stranded (ds) RNA-dependent enzymes, protein kinase (PKR), and 2-5A-synthetase/RNase L (referred to as the 2-5A system) might be a mechanism mediating anticellular and antiviral actions of interferon (i.f.n.). To counteract the effect of i.f.n., animal viruses have acquired genes that block specific i.f.n. pathways. Among poxviruses, vaccinia virus (VV) encodes E3L, a dsRNA-binding protein, which inhibits activation of i.f.n.-induced PKR. It has been proposed that E3L might also block activation of the 2-5A system, but direct proof is lacking. To establish if E3L inhibits the 2-5A system, we have developed a method to assay apoptosis induced by increased production of enzymes in the 2-5A pathway, as well as of their putative modulators. This assay is based on the use of cells derived from homozygous PKR knockout mice (Pkr-/-) infected with a VV mutant lacking E3L (delta E3L) and transiently transfected with a luciferase reporter gene together with plasmid vectors expressing 2-5A-synthetase, RNase L, or E3L, all controlled by the same inducible promoter. We found that expression of 2-5A-synthetase inhibited luciferase activity in a dose-response manner, reaching inhibition values of 80% relative to transfections with control plasmids. Similar results were obtained by transfection with an RNase L vector, although in this case the extent of inhibition was further enhanced upon coexpression of 2-5A-synthetase and RNase L. Inhibition of protein synthesis mediated by the 2-5A system correlated well with induction of apoptosis. Transfection of cells with a plasmid vector expressing E3L together with 2-5A-synthetase completely prevented apoptosis induced by this enzyme. We conclude that VV E3L acts as an inhibitor of the i.f.n.-induced 2-5A-synthetase enzyme.

Bcl-2 prevents nitric oxide-mediated apoptosis and poly(ADP-ribose) polymerase cleavage.

Toxic effects of nitric oxide (NO) were suggested to be mediated by its metabolite peroxynitrite, a strong oxidizing agent. To determine if antioxidative effects of Bcl-2 protooncogene can prevent NO-mediated apoptosis, we used vaccinia virus recombinants expressing mouse inducible NO-synthase, iNOS, or human bcl-2 genes. Expression of iNOS in HeLa G cells induces apoptosis which can be prevented by co-expression of bcl-2 or by addition of reduced glutathione or N-acetylcysteine. We demonstrate that this NO-induced apoptosis proceeds through the activation of interleukin-1 beta-converting enzyme-like proteases and cleavage of the poly(ADP-ribose) polymerase, an effect which is also prevented by Bcl-2.

Inhibition of vaccinia virus DNA replication by inducible expression of nitric oxide synthase.

Nitric oxide (NO) exerts multiple biologic roles in animal cells through differential regulation of three distinct forms of NO synthase encoded by separate genes. Macrophage-inducible nitric oxide synthase (iNOS) has been correlated with inhibition of viral growth, but little is known about the mechanism of this effect. To study the antiviral role of NO, we have generated a vaccinia virus (VV) recombinant expressing iNOS under the control of Escherichia coli LacI operator/repressor elements. When cultured cells of various origins are infected with this recombinant virus, there is inducible expression of iNOS in the presence of isopropylthio-beta-galactoside, as determined by Western blot and by detection of nitrite, a NO oxidation product. The levels of nitrite increase with time after infection, correlating with marked inhibition of VV DNA and late protein synthesis. Expression of VV early proteins is not affected by NO. Inhibition of VV DNA synthesis is likely to be in part a consequence of NO-mediated inhibition of viral ribonucleotide reductase, as this inhibition can be partially overcome by addition of deoxyribonucleosides. Inhibition of the essential viral functions by NO results in a reduction of virus yields by 50 to 90%, depending on the cell line. Thus, our results demonstrate a direct antiviral effect of NO, with inhibition of VV replication occurring at the level of DNA synthesis.

Interferon-gamma severely inhibits DNA synthesis of vaccinia virus in a macrophage cell line.

Interferon-gamma inhibits the growth of several viruses; however, the exact mechanism of this inhibition has not been clearly defined. In this report, we show that in a monocyte/macrophage cell line J774.G8 IFN-gamma inhibits the growth of vaccinia virus (VV) in a dose-dependent manner. This inhibition is due to a block in VV DNA synthesis and correlates with an increased generation of reactive nitrogen intermediates.

The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells.

The role of the interferon-induced double-stranded RNA (dsRNA)-activated human p68 protein kinase as an inhibitor of protein synthesis has been inferred from work with cell-free systems, but direct proof in animal cells is lacking. To document the action of p68 protein kinase in vivo, we have used an infection-transfection system where expression of p68 is driven by a vaccinia virus promoter regulated by the lacl repressor/operator controlling elements. In cultured cells infected with vaccinia virus and transfected with a plasmid containing the p68 gene, there is synthesis of p68 when lacl repressor is inhibited with isopropyl-beta-D-thiogalactoside. When infection-transfections are carried out with the p68 gene together with the luciferase (LUC) reporter gene, a strong inhibition of LUC expression developed with time postinfection. This inhibition was not observed with a mutant form of the kinase (Lys-->Arg at position 296) and it was reversed by antisense expression of the p68 gene. During inhibition of LUC expression the protein kinase was phosphorylated, possibly as a result of autophosphorylation activated by the dsRNA forms which are known to accumulate in vaccinia virus-infected cells. Inhibition of LUC expression was at the level of translation. Our findings demonstrate that expression and activation of the human p68 protein kinase in vivo potently inhibits protein synthesis.

First demonstration of porphyrins in the lacrimal glands: similarity to the Harderian gland.

The Harderian gland of rodents is the only known tissue with physiological occurrence of high concentrations of porphyrins. In this report we describe the occurrence of considerable concentrations of porphyrins in the extra-orbital and intra-orbital lacrimal glands of the male rat. Similarly as in the Harderian gland, HPLC analysis revealed protoporphyrin as being the prevalent porphyrin homologue in the lacrimal glands. The results may contribute to elucidation of the yet unknown function of the Harderian gland and its porphyrins.