Background: Determination of mitochondrial membrane potential (ΔΨ_m) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of ΔΨ_m induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis.Methods: ΔΨ_m in BSC‐40 and HeLa G cell lines was determined by flow cytometry and spectrofluorometry. Its changes induced by specific mitochondrial inhibitors were evaluated using 3,3′‐dihexyloxacarbocyanine iodide (DiOC₆(3)), tetramethylrhodamine ethyl ester, and MitoTracker Red. Mitochondrial function was further characterized by oxygen consumption.Results: Inhibition of respiration by antimycin A or uncoupling of mitochondria by FCCP decreased ΔΨ_m in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of ΔΨ_m in HeLa G cells and an increase of ΔΨ_m in BSC‐40 cells. Statistically significant differences in ΔΨ_m between the two cell lines were found with both flow cytometry and spectrofluorometry. Respirometry showed higher basal and FCCP‐stimulated respiration in BSC‐40 cells.Conclusion: Changes of ΔΨ_m and oxygen consumption showed that BSC‐40 cells are more sensitive than HeLa G cells to inhibitors of mitochondrial function, suggesting that BSC‐40 cells are more dependent than HeLa G cells on aerobic ATP production. Determination of ΔΨ_m changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry. Cytometry Part A 52A:110–116, 2003.

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.

Vaccinia virus (VV) infects a broad range of host cells, and while it usually causes their lysis (i.e. necrosis), the nature of the cell-death phenomenon is not well understood. In this study, we show that VV induces apoptosis of cells of the murine macrophage line J774.G8, as revealed by morphological signs, DNA ladder formation, changes of mitochondrial membrane potential and annexin-V positivity. Apoptosis occurred in both untreated and IFN-γ-pretreated macrophages, and could not be inhibited by aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase. Inhibition of VV DNA synthesis and late gene expression by cytosine arabinoside also did not prevent apoptosis, while heat- or psoralen/UV-inactivated VV did not cause any apoptosis. Thus, VV early gene expression seems to be required for induction of apoptosis. At the cellular level, infection with VV induced a decrease in the levels of Bcl-x_L, an anti-apoptotic member of the Bcl-2 family. The importance of loss of Bcl-x_Lwas demonstrated by prevention of VV-mediated apoptosis on expression of Bcl-2, a functional homologue of Bcl-x_L. Our findings provide evidence that induction of apoptosis by VV in macrophages requires virus early gene expression, does not involve nitric oxide, induces a decrease in mitochondrial membrane potential and is associated with altered levels of Bcl-x_L.

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.