Modification of osteopontin and MMP-9 levels in patients with psoriasis on anti-TNF-α therapy.

Psoriasis is a chronic skin inflammatory disease in which a pleiotropic cytokine, tumor necrosis factor alpha (TNF-α), plays a central role, as demonstrated by the clinical success of anti-TNF-α therapy. Among the multiple effects of TNF-α on keratinocytes, the induction of matrix metalloproteinase-9 (MMP-9), a collagenase implicated in joint inflammation, might be one of the key mechanisms in psoriasis pathogenesis. Interestingly, MMP-9 expression can be enhanced also by osteopontin (OPN), a glycosylated protein whose levels are increased in skin and peripheral blood mononuclear cells (PBMC) of psoriasis patients. The aim of the current study is to investigate the relationship between OPN, MMP-9 and TNF-α in psoriasis. Our survey identified high levels of both OPN and MMP-9 in PBMC as well as skin of psoriatic patients with respect to healthy controls. Significant reduction of OPN and MMP-9 levels in PBMC, plasma and lesional skin of psoriasis patients was observed after 24 weeks of anti-TNF-α therapy. Moreover, OPN and MMP-9 were enhanced by TNF-α and down-regulated by anti-TNF-α treatment in healthy PBMC. These findings may suggest that OPN and MMP-9 may be regulated by TNF-α, indicating a possible role in the pathogenesis of psoriasis.

3-O-methylfunicone, from Penicillium pinophilum, is a selective inhibitor of breast cancer stem cells.

OBJECTIVES: Cancer stem cells make up a subpopulation of cells within tumours that drive tumour initiation, growth and recurrence. They are resistant to many current types of cancer treatment, causing failure of such therapeutic approaches, including chemotherapy and radiotherapy. In the study described here, anti-proliferative effects of 3-O-methylfunicone (OMF), a metabolite from Penicillium pinophilum, were investigated on human breast cancer MCF-7 cells and cancer stem cells selected as mammospheres derived from MCF-7s. MATERIALS AND METHODS: Stemness markers were analysed on isolated mammospheres showing positive expression of CD24, CD29, CD44, CD133, CD184 and CD338. Cell proliferation and apoptosis were analysed by flow cytometry and RT-PCR. Cell colony formation assays were performed to evaluate colony formation of mammospheres. RESULTS AND CONCLUSION: OMF treatment affected both MCF-7 and mammosphere growth, inducing apoptosis. In addition, OMF strongly reduced stemness markers and survivin, hTERT and Nanog-1 gene expression. Growth of colonies in soft-agar was significantly affected by OMF treatment, too. Lastly, we tested ability of MCF-7 cells to form mammospheres after treatment with OMF or cisplatin, demonstrating that OMF treatment resulted in drastic reduction in number of mammospheres. These results introduce OMF as an effective molecule in suppressing breast cancer stem cells.

Antibiotic susceptibility of respiratory pathogens recently isolated in Italy: focus on cefditoren.

The aim of this study was to evaluate the in vitro antibiotic susceptibility of respiratory pathogens recently isolated in Italy to commonly used antibiotics including cefditoren. Six clinical microbiological laboratories collected, between January and September 2009, a total of 2,510 respiratory pathogens from subjects with community-acquired respiratory tract infections (CARTI). Ceftditoren, out of all the beta-lactams studied, had the lowest MIC(90 )against 965 strains of Streptococcus pneumoniae examined, followed by cefotaxime and ceftriaxone (2% resistance in penicillin-resistant S. pneumoniae (PRSP)). Against 470 Haemophilus influenzae , independently of their production of beta-lactamases or ampicillin resistance, cefditoren was the oral cephalosporin with the best in vitro activity, comparable to that of the injectable cephalosporins and levofloxacin. Higher MIC(90)s were found for the macrolides (4 - 16 mg/l) and cefaclor (4 - 32 mg/l). As was foreseeable, Streptococcus pyogenes (225 strains) was uniformly sensitive to all the beta-lactam antibiotics, but the elevated MIC(90 )values reduced (<75%) susceptibility of this pathogen to macrolides. Beta-lactamase-negative Moraxella catarrhalis (100 strains) had reduced susceptibility only to the macrolides, while the 250 beta-lactamase-producing strains also had reduced susceptibility to cefuroxime. Levofloxacin showed the lowest MIC(50)/MIC(90 )values in the producing strains, whereas cefditoren, cefotaxime and ceftriaxone in the non-producers. As regards the enterobacteriaceae, cefditoren and levofloxacin had the lowest MIC(90)s against Klebsiella pneumoniae. Cefditoren and the third-generation injectable cephalosporins had the lowest MIC(90)s against Escherichia coli (100% susceptibility) while levofloxacin was less active (86% susceptibility).In conclusion, cefditoren's wide spectrum and high intrinsic activity, as well as its capacity to overcome most of the resistance that has become consolidated in some classes of antibiotics widely used as empiric therapy for CARTI, allows us to suggest that cefditoren might be included in the european guidelines as one of the first-choice antibiotics in the treatment of CARTI.

3-O-Methylfunicone, a metabolite produced by Penicillium pinophilum, modulates ERK1/2 activity, affecting cell motility of human mesothelioma cells.

OBJECTIVES: 3-O-methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility in a variety of human solid tumours. The aim of this study was to demonstrate whether OMF has the ability to arrest cell division and motility, in a human mesothelioma cell line. Malignant mesothelioma is an aggressive cancer that does not respond to standard therapies the cells of which are considered to be highly resistant to apoptosis. MATERIAL AND METHODS: Cell motility and invasion were measured using a modified Boyden chamber. Gene expression was examined by RT-PCR, while ERK1/2 was investigated by Western blot analysis. All experiments were also performed on primary cultures of mesothelial cells. RESULTS: The present study shows that OMF inhibited motility of the NCI mesothelioma cell line by modulating ERK signalling activity, and affected alphaVbeta5 integrin and MMP-2 expression, inducing marked downregulation at both mRNA and protein levels. Substantial downregulation of VEGF gene expression was also demonstrated. These effects were not observed in normal mesothelial cell cultures. CONCLUSION: OMF may have potential as a naturally derived anti-tumour drug for treatment of mesothelioma.

Effect of temperature on the shift of Pseudomonas fluorescens from an environmental microorganism to a potential human pathogen.

Pseudomonas fluorescens is a Gram-negative bacterium generally considered of scarce clinical significance. However, in the last few years, the isolation of P. fluorescens as the causative agent of nosocomial infections has rapidly increased. P. fluorescens is a psychrophile microorganism which grows at an optimal temperature of 25-30 degrees Celcius. In spite of this constraint, it has recently been reported that the human physiological temperature does not appear to be a barrier for this microorganism. In this study we examined the ability of P. fluorescens, grown at 28 degrees C or at 37 degrees C, to adhere to cultured human A549 pulmonary cells and to form biofilm. The ability of P. fluorescens to induce expression of proinflammatory cytokines, beta-defensin 2 and the intercellular adhesion molecule-1 was also investigated. Our results clearly indicate that inflammatory mediators are induced when the microorganism is grown at a lower temperature, while biofilm is formed only at 37 degrees C. The results presented are consistent with previous reports indicating P. fluorescens as an opportunistic pathogen and underscore the urgent need for further studies to better characterize the virulence of this microorganism.

Effects of toluidine blue-mediated photodynamic therapy on periopathogens and periodontal biofilm: in vitro evaluation.

Photodynamic therapy (PDT) is a selective modality of killing targeted cells, mostly known for its application in neoplasms. PDT can be considered to be an alternative method for the elimination of periodontal bacteria from the pocket without harms for the resident tissues. Therefore, PDT may replace systemic antibiotics and enhance the effect of mechanical treatments of periodontal defects. This effort focused on the in vitro sensitization of periopathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia ) Toluidine Blue mediated and on the use of a Diode laser emitting source. The objective of this research was to evaluate the bactericidal in vitro effect of laser diodes 830 nm (as the light source) after photosensitization with Toluidine Blue (TBO) on the following periopathogenic bacteria: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia. After evaluating the effect on the single bacterial strain, the ability of Diode Laser to disrupt the structure of biofilms produced by A. actinomycetemcomitans after photosensitization with TBO was also analyzed. The study suggests that the association of TBO and diode laser light 830 nm is effective for the killing of bacteria strains and determines the photoinactivation of Aggregatibacter biofilms. In summary, photodynamic therapy has effectively shown its capabilities and, therefore, it can be considered a valid alternative approach to antimicrobial therapy of periodontitis.

Hepatocyte growth factor (HGF) modulates Leydig cell extracellular matrix components.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays multiple roles during mammalian development. We previously demonstrated that in the postnatal testes, the HGF receptor, c-met, is expressed by Leydig cells and HGF increases the steroidogenetic activity of the cells. In the present article, we report that HGF modifies the composition of the extracellular matrix of cultured Leydig cells. We show that HGF increases the metabolic activity of isolated Leydig cells; in particular, the factor increases urokinase plasminogen activator and matrix metalloproteinase 2 secretion. We have also shown that the levels of active transforming growth factor beta are increased by HGF. On the contrary, using the Western blotting technique, a strong reduction in the amount of fibronectin present in the culture medium of cells cultured in the presence of HGF has been detected. The presented data demonstrate that HGF modulates several functional activities of Leydig cells, further supporting the hypothesis that this factor has a relevant role in the regulation of mammalian spermatogenesis.

3-O-methylfunicone, a metabolite of Penicillium pinophilum, inhibits proliferation of human melanoma cells by causing G(2) + M arrest and inducing apoptosis.

OBJECTIVES: Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3-O-methylfunicone (OMF), a metabolite of Penicillium pinophilum, has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines. MATERIALS AND METHODS: Cell proliferation and apoptosis were analysed by flow cytometry, DNA fragmentation, caspase-3 and caspase-9 activation, and PARP-1 cleavage. RESULTS: We demonstrated that OMF affected cell proliferation in a time- and dose-dependent manner, reaching the best effect at concentration of 80 microg/ml for 24 h. Flow cytometry revealed that OMF caused significant G(2) phase arrest, which was associated with marked decrease in cyclin B1/p34(cdc2) complex and p21 induction. OMF also induced marked decrease of survivin expression. Reduced levels of apoptosis were evident after silencing p21 expression in both cell lines. Finally, the effect exercised by OMF on hTERT and TEP-1 gene expression confirmed the ability of this molecule to interfere with replicative ability of cells. CONCLUSIONS: The results reported here seem to suggest that OMF as a promising molecule to include in strategies for treatment of melanoma.

Antibacterial activity of cefditoren against major community-acquired respiratory pathogens recently isolated in Italy.

In this study we evaluated the in vitro activities of cefditoren--a broad-spectrum oral cephalosporin--and other comparator agents against 2,396 fresh isolates from community-acquired respiratory tract infections, collected from 6 clinical Italian microbiology laboratories. On penicillin-susceptible pneumococci and Streptococcus pyogenes, cefditoren demonstrated to be the most active antibiotic (MIC(90)values of 0.03 and 0.06 mg/L respectively), showing only a slight decrease in potency on penicillin-intermediate and resistant pneumococci (MIC(90)value 0.5 mg/L, 1.0 mg/L respectively). All the other comparators displayed MIC(90 )values of 4 - 8 mg/L for penicillins and of 4 to >64 mg/L for the oral cephalosporins. Cefditoren and levofloxacin were the most active against MSSA (MIC(90)0.5 mg/mL). Cefditoren displayed a uniformly potent inhibitory activity (MIC(90)of 0.03 mg/L) against all strains of Haemophilus influenzae, regardless of their ampicillin resistance (mediated or not by beta-lactamase production), while against Moraxella catarrhalis MIC(90)values were higher against beta-lactamase-positive (0.25 mg/L). Cefditoren was active also against Klebsiella pneumoniae and Escherichia coli : in this case its activity was comparable with that of levofloxacin. In conclusion, cefditoren, due to its potent activity, is a new effective therapeutic option for the treatment of respiratory tract infections.

Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates.

OBJECTIVES: Helicobacter pylori infection is a major health problem worldwide, and effective eradication of the infection is mandatory. The efficacy of recommended eradication regimens is approximately 70%. To avoid treatment failure and the consequent development of secondary resistance(s), it is important to choose the most appropriate first-line treatment regimen. This choice should also be made based on the knowledge of the antimicrobial resistance peculiar to a given geographical area. We evaluated the prevalence of antimicrobial-resistant H pylori strains isolated from naive patients and from patients with previous unsuccessful treatments. METHODS: This study examined 109 H pylori-infected subjects (Group 1) who had never received an eradication treatment and 104 H pylori-infected subjects (Group 2) who had failed one or more eradication treatments. Resistance to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET) and levofloxacin (LEV) was determined using the epsilometer test. The significance of differences was evaluated by the chi2 test. RESULTS: The prevalence of antimicrobial resistance was 0% versus 3.1% to AMO, 0% versus 2% to TET, 27% versus 41.3% to MET (p<0.05), 18% versus 45.8% to CLA (p<0.05) and 3% versus 14.6% to LEV (p<0.05) in Group 1 vs Group 2, respectively. In Group 2, there was an increased prevalence of H pylori strains resistant to multiple antimicrobials. CONCLUSIONS: This study confirms the high prevalence of H pylori strains resistant to CLA and MET, and indicates that unsuccessful treatments significantly increase resistance. Choosing eradication regimens other than standard triple therapy as a first-line therapy should be advisable in areas with high primary antimicrobial resistance prevalence.

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells.

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin-associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co-transfected with CagA or HspB. Moreover, VacA over-expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor-mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co-operate to execute cell death with a prevalence of the mitochondrial pathways. Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of human gastric epithelial cells.

Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells.

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.

Immunomodulatory effects of peptide T on human keratinocyte cells.

BACKGROUND: Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. PT is from the V2 region of HIV-1 gp120, an exterior envelope glycoprotein that is a target for host immune responses. The anti-inflammatory mechanisms of PT are not well understood. OBJECTIVES: We studied the immunomodulatory effects of PT on the human keratinocyte cells. METHODS: Cultured human keratinocytes were treated with PT, proteins extracted and analysed by Western blotting and reverse transcriptase polymerase chain reaction. RESULTS: Our findings show reduced expression of intercellular adhesion molecule 1 and an increase in transforming growth factor (TGF)-beta and heat shock protein (HSP)-70 in human keratinocyte cells treated with PT. The HSP-70 increase is modulated by TGF-beta. In fact, we demonstrated that anti-TGF-beta antibodies reduce HSP-70 overexpression. In addition, we show a modulation of alphav integrins after 4 hours of treatment with PT. These receptors favour keratinocyte migration and epidermal regeneration. It has been reported that overexpression of HSP results in dramatic changes to intermediate filaments. These proteins act on keratin intermediate filaments and determine their retraction. The consequence is cell-cell contact detachment and inhibition of cellular hyperproliferation. CONCLUSIONS: Our results support the beneficial effect of PT found in vivo, suggesting, moreover, the primary role of keratinocytes upon which PT acts directly by stimulating the anti-inflammatory function and favouring the regeneration of tissue.

Antimicrobial action of Nitens mouthwash (cetyltrimethylammonium naproxenate) on multiple isolates of pharyngeal microbes: a controlled study against chlorhexidine, benzydamine, hexetidine, amoxicillin, amoxicillin-clavulanate, clarithromycin, and cefaclor.

BACKGROUND: Acute oropharyngeal and respiratory tract infections are due to a wide spectrum of microorganisms. The aim of this study was to compare and evaluate the in vitro activity of four antiseptics (cetyltrimethylammonium naproxenate, chlorhexidine, benzydamine, hexetidine) to four antibiotics (amoxicillin, amoxicillin-clavulanate, clarithromycin, cefaclor) on strains of Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes and Streptococcus pneumoniae. METHODS: Susceptibility tests were performed on 90, aerobic and anaerobic, bacterial strains, isolated from nasopharyngeal swabs and sputum. Minimum inhibitory concentrations (by microdilution) and minimum bactericidal concentrations were determined and compared. RESULTS: Our selected panel of bacteria was highly susceptible to the antiseptics, particularly to chlorhexidine and naproxenate, even more so than two of the most frequently used antibiotics. Data were statistically significant (p < 0.005). CONCLUSIONS: In view of their bactericidal and anti-inflammatory properties, these antiseptics may be effective in controlling the transitory colonization of the oral cavity by microbes that cause or worsen disease in patients with mild infections.

Isolated autoimmune response: definition, analysis of the prevalence in an outpatient rheumatology clinic, relationship to pre-clinical autoimmune disease and infections by hepatotropic viruses.

OBJECTIVE: To define the clinical significance of non-organ specific autoantibody positivity in patients in whom routine clinical and laboratory examinations did not detect any disease that might have caused the serological finding. METHODS: Out of 1,120 patients consecutively admitted to an outpatient rheumatology clinic, 28 were referred for the evaluation of an autoantibody positivity unrelated to the clinical status. These patients and 28 sex- and age-matched controls underwent a specific work-up with the aim of detecting any underlying infection or autoimmune disease. RESULTS: Eight of the 28 patients (28.5%) were found to be affected by a previously undetected disease: 3 chronic hepatitis C, 3 Sjögren's syndrome, and 2 autoimmune thyroiditis. The remaining 20 did not show any autoimmune or hepatic disease, although 4 of them showed active infection by HBV (n = 1) or HGV (n = 3) and 15 had had a previous infection by hepatotropic viruses (HBV, CMV or EBV). After a follow-up lasting 6-54 months, none of the last 20 patients developed any autoimmune or chronic hepatic disease. CONCLUSIONS: A diagnostic work-up is necessary in patients presenting with unexpected autoantibody positivity in order to detect an underlying pre-clinical autoimmune disease and/or unexpected hepatic infection. Patients in whom such a work-up fails to point out any condition should be further followed in order to make an early diagnosis of autoimmune disease.

Porins from Salmonella typhimurium accelerate human blood coagulation in vitro by selective stimulation of thrombin activity: implications in septic shock DIC pathogenesis.

The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium, on human blood coagulation was investigated. It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro. Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin. A possible binding of porins with thrombin has been suggested to be the basis of this effect. The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.

Pulmonary disposition of lomefloxacin in patients with acute exacerbation of chronic obstructive pulmonary disease. A multiple-dose study.

In this study we have measured the concentrations of lomefloxacin at steady state in serum and in the intrapulmonary region at specified intervals for 24 h following administration of the last dose of drug in patients suffering from acute exacerbation of chronic obstructive pulmonary disease (COPD). Twenty subjects were enrolled. They received lomefloxacin 400 mg orally once-daily for 5 consecutive days. All patients were divided into five groups, with 4 subjects in each group, according to sampling times (2, 4, 8, 12, and 24 h after the last dose). At bronchoscopy, bronchial biopsies and bronchoalveolar lavage (BAL) were performed. At 12 h after the last dose, serum concentration of lomefloxacin was >1.0 microg/mL and at 24 h it was still detectable, but, at all times, the concentrations in bronchial secretion, bronchial mucosa, and epithelial lining fluid (ELF) were greater than the concentrations in serum [bronchial secretions (pg/mL) = 2.5+/-1.2; 2.2+/-1.0: 2.0+/-1.1; 1.8+/-1.1; 0.6+/-0.3. bronchial mucosa (microg/g) = 5.9+/-2.1; 6.2+/-1.8; 2.6+/-2.2; 1.9+/-1.5; 1.0+/-0.9. ELF (microg/mL) = 6.9+/-2.8; 5.9+/-2.6; 3.1+/-1.9; 2.2+/-1.0; 0.8+/-1.3. serum (microg/mL) = 3.2+/-1.4; 2.8+/-0.9: 2.1+/-1.5; 1.2+/-1.1; 0.4+/-0.81. We must stress that we observed a large inter-individual variability in concentrations. Our data show that lomefloxacin once-daily induces high and sustained concentrations in the various potential sites of pulmonary infection and clearly indicate that the pharmacokinetic behavior of this fluoroquinolone permits once-daily administration in patients with acute exacerbations of COPD.

UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins.

It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells.