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Protein S-glutathionylation, the reversible binding of glutathione to protein thiols (PSH), is involved in protein redox regulation, storage of glutathione, and protection of PSH from irreversible oxidation. S-Glutathionylated protein (PSSG) can result from thiol/disulfide exchange between PSH and GSSG or PSSG; direct interaction between partially oxidized PSH and GSH; reactions between PSH and S-nitrosothiols, oxidized forms of GSH, or glutathione thiyl radical. Indeed, thiol/disulfide exchange is an unlikely intracellular mechanism for S-glutathionylation, because of the redox potential of most Cys residues and the GSSG export by most cells as a protective mechanism against oxidative stress. S-Glutathionylation can be reversed, following restoration of a reducing GSH/GSSG ratio, in an enzyme-dependent or -independent manner. Currently, definite evidence of protein S-glutathionylation has been clearly demonstrated in few human diseases. In aging human lenses, protein S-glutathionylation increases; during cataractogenesis, some of lens proteins, including alpha- and beta-crystallins, form both mixed disulfides and disulfide-cross-linked aggregates, which increase with cataract severity. The correlation of lens nuclear color and opalescence intensity with protein S-glutathionylation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses. Recently, specific PSSG have been identified in the inferior parietal lobule in Alzheimer's disease. However, much investigation is needed to clarify the actual involvement of protein S-glutathionylation in many human diseases.

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

Epidermis and keratinizing oral mucosa (KOM) are effective barriers against a wide spectrum of insults. The optimal form of protection provided by each epithelium is determined also by the molecular composition of desmosomes. Up to now, the expression of the "skin type" desmosomal cadherins, i.e. desmocollin 1 (Dsc1) and desmoglein 1 (Dsg1), was correlated with the morphological features of keratinocyte terminal differentiation in epidermis, but not in KOM. The aim of the present study was to investigate Dsc1 and Dsg1 expression in adult human KOM compared to epidermis. Biopsies of epidermis and KOM were obtained from young healthy adults (n=6) and simultaneously processed for immunofluorescence analysis, post-embedding immunogold electron microscopy (immunogold EM), and RT-PCR analysis. For molecular biology analysis, as a negative control, we considered human fibroblasts. By immunofluorescence and immunogold EM, Dsc1 labeling was not detected in any suprabasal layer of KOM, but it was present in the upper spinous/granular layers of epidermis. Immunofluorescence and transmission electron microscopy analysis showed that (i) Dsg1 expression was evident in the spinous, granular, and horny layer of the oral epithelium and (ii) Dsg1 immunoreactivity was always lower in desmosomes between oral keratinocytes than in all epidermal junctions. RT-PCR analysis confirmed that in KOM Dsc1 gene expression was undetectable. On the whole, these observations suggest a weakened adhesion in KOM, allowing oral keratinocytes to undergo a faster transition throughout the living layers of the epithelium. The intrinsic and specific regulation of the molecular composition of desmosomes can contribute in defining a specific keratinocyte phenotype in KOM and in epidermis.

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.

Background: The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover., Methods: Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR)., Results: Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment., Conclusions: Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

We characterized the effect of chronic ochratoxin A (OTA) on rat kidney cortex, analyzing collagen content and collagen turnover and the major markers of epithelial-to-mesenchymal transition (EMT), such as alpha-smooth muscle actin (alphaSMA), cadherins, and MMP-9. Because OTA nephrotoxicity is mediated by free radicals, we also investigated whether antioxidants in red wine provided protection for the kidney and attenuated OTA-induced EMT. Collagen content, determined by computerized analysis of Sirius red-stained kidney sections, increased in OTA, OTA-wine, and OTA-EtOH treated rats. In kidney cortex homogenates, COL-I and COL-III mRNA levels tended to rise in OTA treated rats, but were similar to CT after OTA-wine and OTA-EtOH administration. TIMP-1 gene expression was up-regulated in OTA, OTA-wine, and OTA-EtOH treated rats. LH2b mRNA/COL-I mRNA was significantly up-regulated in OTA-wine and OTA-EtOH treated rats, compared with CT and OTA alone. TGF-beta1 signaling tended to dominate after OTA, OTA-wine, and OTA-EtOH. MMP-1 protein levels were not affected. OTA induced proMMP-9 and alphaSMA overexpression, decreases of E-cadherin and N-cadherin, and DSC-2 up-regulation. OTA-wine caused a further, unexpected decrease of E- and N-cadherins and further up-regulation of OTA-induced DSC-2, while strongly reducing the OTA-induced increases of alphaSMA and proMMP-9. Posttranslational collagen modifications, such as decreased collagen degradation through MMP inhibition and increased collagen cross-links, seem to be key mechanisms leading to OTA-induced kidney cortex fibrosis. This mechanism was not affected by red wine in these conditions. Red wine seems to have some protective role against OTA-induced EMT, although without completely blocking the process and determining a condition in which abundant cells display an intermediate translational phenotype, but there are no alphaSMA or epithelial markers.

Objective: To evaluate mast cell (MC) density, in liver tissues taken from young and aging rats treated with carbon tetrachloride (CCl4) or untreated, as a quantitative marker of acute liver inflammation and to investigate whether the density of MCs varied with the rats' age., Study Design: Rats aged 2, 6, 12 and 19 months treated intraperitoneally with CCl4 were killed 2 and 24 hours after intoxication. Hepatocellular damage was established by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Four histologic sections of 12 specimens from each age group were stained with toluidine blue to identify the MCs, which were counted using a computer-assisted image analysis system., Results: Histology showed hepatocellular necrosis with inflammatory infiltration both 2 and 24 hours after intoxication. Serum AST levels were high in the 6- and 12-month-old rats, whereas ALT levels were high in the those aged 2 and 19 months. Two and 24 hours after intoxication, MC density increased considerably in young rats but less so in rats aged 19 months., Conclusion: MC density can be a useful marker of acute liver inflammation. The greater density in young rats suggests that older rats have a reduced immune response or recruit fewer MCs.