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Background: This single-centered randomized controlled clinical trial aimed to evaluate the effectiveness of micro-osteoperforations (MOPs) in accelerating the orthodontic retraction of maxillary incisors. Methods: Forty-two patients aged 16–40 were recruited and randomly assigned into two groups, one which underwent MOPs (MOPG) in the buccal and palatal region of all maxillary incisors immediately before the start of retraction and one which did not (CG). Eligibility criteria included the orthodontic need for maxillary first premolars extraction and space closure in two phases. The primary outcome of the study consisted of measuring the rate of space closure and, consequently, the rate of incisors’ retraction using digital model superimposition 14 days later and monthly thereafter for the next 4 months. The secondary outcomes included measuring anchorage loss, central incisors’ inclination, and root length shortening, analyzed using cone beam computed tomography scans acquired before retraction and 4 months after retraction. Randomization was performed using QuickCalcs software. While clinical blinding was not possible, the image’s examinator was blinded. Results: Twenty-one patients were randomly assigned to each group. However, due to various reasons, a total of 37 patients (17 male and 20 female) were analyzed (mean age: 24.3 ± 8.1 years in the MOPG; 22.2 ± 4.2 years in the CG) during the trial. No statistically significant difference was found between the MOPG and the CG regarding the incisors’ retraction measured at different time points at the incisal border (14 days, 0.4 mm vs. 0.5 mm; 1 month, 0.79 mm vs. 0.77 mm; 2 months, 1.47 mm vs. 1.41 mm; 3 months, 2.09 mm vs. 1.88 mm; 4 months, 2.62 mm vs. 2.29 mm) and at the cervical level (14 days, 0.28 mm vs. 0.30 mm; 1 month, 0.41 mm vs. 0.32 mm; 2 months, 0.89 mm vs. 0.61 mm; 3 months, 1.36 mm vs. 1.10 mm; 4 months, 1.73 mm vs. 1.39 mm). Similarly, no statistically significant differences were detected in the space closure, anchorage loss, central incisors’ inclination, and radicular length between groups. No adverse effect was observed during the trial. Conclusions: MOPs did not accelerate the retraction of the maxillary incisors, nor were they associated with greater incisor inclination or root resorption.

Purpose: Patients with functional hypothalamic amenorrhea (FHA) could commonly have bone damage, often preceded by metabolic alterations due to a relative energy deficit state. To date, there are no markers capable of predicting osteopenia before it is manifested on DXA. Irisin is a myokine that promotes the differentiation of osteoblastic cells and appears to be inversely correlated with the incidence of bone fragility and fractures in postmenopausal women. The aim of this study was to measure irisin levels in FHA patients and to correlate it with bone density parameters. Methods: Thirty-two patients with FHA and 19 matched controls underwent the same clinical and laboratory evaluation. Results: Irisin and body mass index (BMI) were significantly lower in the case group than in healthy controls (2.03 ± 0.12 vs. 2.42 ± 0.09 p< 0.05 and 19.43 ± 2.26 vs. 22.72 ± 0.67 p< 0.05, respectively). Additionally, total body mass density (BMD g/cm2) was significantly lower in the case group than in the healthy controls (1.09 ± 0.08 vs. 1.14 ± 0.05, p< 0.05), without signs of osteopenia. Conclusions: The FHA group showed lower irisin levels associated with significantly reduced BMD parameters that did not reach the severity of osteopenia. Therefore, we could speculate that irisin could predict DXA results in assessing modifications of body composition parameters. Future research is warranted to study these parameters in a larger population to confirm our results, so that irisin could be used as a predictor and screening method for bone deprivation. Furthermore, irisin is strictly related to energy metabolism and could be an indirect marker of nutritional status in FHA patients, identifying earlier states of energy deficit.

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.

Selenium is a trace element, nutritionally classified as an essential micronutrient, involved in maintaining the correct function of several enzymes incorporating the selenocysteine residue, namely the selenoproteins. The human selenoproteome including 25 proteins is extensively described here. The most relevant selenoproteins, including glutathione peroxidases, thioredoxin reductases and iodothyronine deiodinases are required for the proper cellular redox homeostasis as well as for the correct thyroid function, thus preventing oxidative stress and related diseases. This review summarizes the main advances on oxidative stress with a focus on selenium metabolism and transport. Moreover, thyroid-related disorders are discussed, considering that the thyroid gland contains the highest selenium amount per gram of tissue, also for future possible therapeutic implication.

Imaging studies have hystorically been used to support the clinical otorhinolaryngological evaluation of the upper respiratory tract for the diagnosis of obstructive causes of oral breathing.

Topoisomerases are ubiquitous enzymes involved in maintaining genomic stability of the cell by regulating the over- or underwinding of DNA strands. Besides their customary functions, topoisomerases are important cellular targets of widely used anticancer drugs. In particular, topoisomerase IIα (Top2α) has been postulated as the primary molecular target of anthracycline’s anticancer activity, whereas topoisomerase IIβ (Top2β), the only Top2 present in heart tissue, seems to be involved in the development of anthracycline-induced cardiotoxicity. Noteworthy, cardiotoxicity is the most frequent adverse effect of both conventional and modern anticancer targeted therapy, representing the leading noncancer-related cause of morbidity and mortality in long-term survivors. The molecular mechanisms of anthracyclineinduced cardiotoxicity have been investigated for decades and, despite the numerous mechanistic hypotheses put forward, its aetiology and pathogenesis still remain controversial. This review is aimed at focusing on the double edge sword of topoisomerase-anthracycline interaction, and, in particular, on the potential role of topoisomerases in anthracyclines anticancer activity as well as in the pathogenesis of anthracycline-induced cardiotoxicity.

Introduction Infertility affects 50 to 80 million (between 8 and 12% of couples). Male factor is a cause of infertility in almost half of the cases, mainly due to oligoasthenoteratozoospermia. DNA fragmentation is now considered an important factor in the aetiology of male infertility. We studied the effects on semen analysis and on DNA fragmentation of in vivo admnistration of Myo-Inositol and Tribulus Terrestris plus Alga Ecklonia plus Biovis (Tradafertil; Tradapharma Sagl, Swizerland) in men with previously diagnosed male infertility.Materials and Methods Sixty patients were enrolled in the present study and were randomized into two subgroups: the group A who received Myo-inositol 1000 mg, Tribulus Terrestris 300 mg, Alga Ecklonia Bicyclis 200 mg and Biovis one tablet a day for 90 days, and the group B (placebo group) who received one placebo tablet a day for 90 days. The primary efficacy outcome was the improvement of semen characteristics after 3 months’ therapy and the secondary outcome was the reduction of the DNA fragmentation after treatment.Results The groups were homogenous for age, hormonal levels, sperm concentration and all parameters of sperm analysis. Sperm concentration and progressive motility improved after treatment with Tradafertil (3.82 Mil/ml vs. 1.71 Mil/ml; p<0.05; 4.86% vs. 1.00%; p<0.05) as well as the DNA fragmentation (-1.64% vs -0.39%, p<0.001). No side effects were revealed.Conclusions In conclusion, we can affirm that Tradafertil is safe and tolerable. It is a new phytotherapic approach to Oligoasthenoteratospermia (OAT) syndrome that could lead to good results without interacting with hypothalamic–pituitary–gonadal axis.

The clinical efficacy of anthracyclines (e.g., doxorubicin and daunorubicin) in cancer therapy is limited by their severe cardiotoxicity, the etiology of which is still not fully understood. The development of anthracycline-induced cardiomyopathy has been found to correlate with myocardial formation and accumulation of anthracycline secondary alcohol metabolites (e.g., doxorubicinol and daunorubicinol) that are produced by distinct cytosolic NADPH-dependent reductases. The aim of the current study is to identify chemical compounds capable of inhibiting myocardial reductases implied in anthracycline reductive metabolism in an attempt to decrease the production of cardiotoxic C-13 alcohol metabolites. Among the variety of tested compounds (metal chelators, radical scavengers, antioxidants, β-blockers, nitrone spin traps, and lipid-lowering drugs), ebselen, cyclopentenone prostaglandins, nitric oxide donors, and short-chain coenzyme Q analogs resulted in being effective inhibitors of both doxorubicinol and daunorubicinol formation. In particular, ebselen (as well as ebselen diselenide, its storage form in the cells) was the most potent inhibitor of cardiotoxic anthracycline alcohol metabolites with 50% inhibition of doxorubicinol formation at 0.2 mol Eq of ebselen with respect to doxorubicin concentration. The high efficacy, together with its favorable pharmacological profile (low toxicity, lack of adverse effects, and metabolic stability) portends ebselen as a promising cardioprotective agent against anthracycline-induced cardiotoxicity.

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruziat concentrations that did not affect host cell viability. In addition, in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

The clinical efficacy of anthracyclines (e.g., doxorubicin and daunorubicin) in cancer therapy is limited by their severe cardiotoxicity, the etiology of which is still not fully understood. The development of anthracycline-induced cardiomyopathy has been found to correlate with myocardial formation and accumulation of anthracycline secondary alcohol metabolites (e.g., doxorubicinol and daunorubicinol) that are produced by distinct cytosolic NADPH-dependent reductases. The aim of the current study is to identify chemical compounds capable of inhibiting myocardial reductases implied in anthracycline reductive metabolism in an attempt to decrease the production of cardiotoxic C-13 alcohol metabolites. Among the variety of tested compounds (metal chelators, radical scavengers, antioxidants, β-blockers, nitrone spin traps, and lipid-lowering drugs), ebselen, cyclopentenone prostaglandins, nitric oxide donors, and short-chain coenzyme Q analogs resulted in being effective inhibitors of both doxorubicinol and daunorubicinol formation. In particular, ebselen (as well as ebselen diselenide, its storage form in the cells) was the most potent inhibitor of cardiotoxic anthracycline alcohol metabolites with 50% inhibition of doxorubicinol formation at 0.2 mol Eq of ebselen with respect to doxorubicin concentration. The high efficacy, together with its favorable pharmacological profile (low toxicity, lack of adverse effects, and metabolic stability) portends ebselen as a promising cardioprotective agent against anthracycline-induced cardiotoxicity.

Cardiovascular disease (CVD) is the leading cause of death in Western societies and accounts for up to a third of all deaths worldwide. In comparison to the Northern European or other Western countries, the Mediterranean area has lower rates of mortality from cardiovascular diseases and cancer, and this is attributed, at least in part, to the so-called Mediterranean diet, which is rich in plantderived bioactive phytochemicals. Identification of the active constituents of the Mediterranean diet is therefore crucial to the formulation of appropriate dietary guidelines. Lycopene is a natural carotenoid found in tomato, an essential component of the Mediterranean diet, which, although belonging to the carotenoid family, does not have pro-vitamin A activity but many other biochemical functions as an antioxidant scavenger, hypolipaemic agent, inhibitor of pro-inflammatory and pro-thrombotic factors, thus potentially of benefit in CVD. In particular, the review intends to conduct a systematic analysis of the literature (epidemiological studies and interventional trials) in order to critically evaluate the association between lycopene (or tomato products) supplementation and cardiovascular diseases and/or cardiovascular disease risk factors progression, and to prepare provision of evidence-based guidelines for patients and clinicians. Several reports have appeared in support of the role of lycopene in the prevention of CVD, mostly based on epidemiological studies showing a dose-response relationship between lycopene and CVD. A less clear and more complex picture emerges from the interventional trials, where several works have reported conflicting results. Although many aspects of lycopene in vivo metabolism, functions and clinical indications remain to be clarified, supplementation of low doses of lycopene has been already suggested as a preventive measure for contrasting and ameliorating many aspects of CVD.

Anthracyclines are among the most effective anticancer drugs ever developed. Unfortunately, their clinical use is severely limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure, usually refractory to conventional therapy. The pathophysiology of anthracycline-induced cardiomyopathy remains controversial and incompletely understood. The current thinking is that anthracyclines are toxic per se but gain further cardiotoxicity after one-electron reduction with ROS overproduction or two-electron reduction with conversion to C-13 alcohol metabolites. ROS overproduction can probably be held responsible for anthracycline acute cardiotoxicity, but not for all the aspects of progressive cardiomyopathy. Intramyocardial formation of secondary alcohol metabolites might play a key role in promoting the progression of cardiotoxicity toward end-stage cardiomyopathy and congestive heart failure. In this review we also discuss recent developments in: a) the molecular mechanisms underlying anthracycline-induced cardiotoxicity; b) the role of cytosolic NADPH-dependent reductases in anthracycline metabolism; c) the influence of genetic polymorphisms on cardiotoxicity outcome; d) the perspectives on the most promising strategies for limiting or preventing anthracycline-induced cardiotoxicity, focusing on controversial aspects and on recent data regarding analogues of the natural compounds, tumor-targeted formulations and cardioprotective agents.

BACKGROUND Dermoscopic classification of acquired melanocytic nevi (AMN) is based on the evaluation of 3 main criteria—global pattern, pigment distribution, and color. OBJECTIVE To determine whether these features are different in AMN in white people with different skin types (STs) according to the Fitzpatrick classification. DESIGN Digital dermoscopic images of AMN were evaluated, and the correlation of the 3 main dermoscopic criteria with patient ST was analyzed. SETTING Consecutive patients were recruited from 7 pigmented lesion clinics between June 1, 2004, and June 30, 2005. PATIENTS For each patient, the ST (I [always burns, never tans] to IV [rarely burns, tans with ease]) was scored, and 1 representative AMN (defined as the AMN showing a dermoscopic typology that is repeatedly seen in the same patient) was selected and photographed. MAIN OUTCOME MEASURES The distribution of the dermoscopic criteria of AMN in patients with different STs was calculated by univariate analysis. Differences in prevalence were tested using the χ2 test. The correlation between dermoscopic criteria and ST, adjusted for age, sex, and enrolling center, was evaluated by calculating odds ratios and 95% confidence intervals by logistic regression analysis. RESULTS Of 680 included patients, dermoscopic analysis revealed significant differences in the prevalent nevus pattern in the 4 ST groups. Light brown AMN with central hypopigmentation were associated with ST I, and ST IV was associated with the so-called black nevus (P&lt;.001), typified by reticular pattern, central hyperpigmentation, and dark brown coloration. A significant association was also found between multifocal pattern and ST II and ST III. CONCLUSIONS The dermoscopic nevus type varies according to different ST in white people. This knowledge may have an effect on obtaining for biopsy lesions that exhibit unusual dermoscopic patterns when patient ST is considered.Arch Dermatol. 2007;143:351-356--

Antineoplastic therapy with anthracyclines like doxorubicin (DOX) and daunorubicin (DNR) is limited by the possible development of a dose-related cardiomyopathy. Secondary alcohol metabolites like doxorubicinol (DOXol) and daunorubicinol (DNRol), formed by cytoplasmic two-electron reductases, have been implicated as potential mediators of anthracycline-induced cardiomyopathy. In the present study, we characterized the effects of 12 chalcones on the formation of anthracycline secondary alcohol metabolites by rabbit or human heart cytosol and compared them with those of quercetin and other flavonoids. Both chalcones and flavonoids inhibited DOXol or DNRol formation in isolated rabbit heart cytosol. Structure-activity relationships showed that inhibition by chalcones was determined primarily by the position of hydroxyl groups in their phenolic A and B rings. In particular, the presence of a hydroxyl group at C-4‘ in the A ring was an important determinant of the inhibitory activity of chalcones. Among chalcones, 2‘,4‘,2-trihydroxychalcone exhibited the highest inhibition of both DOXol and DRNol formation, but it proved less efficient than quercetin. Different results were obtained with isolated human heart cytosol:? in the latter, 2‘,4‘,2-trihydroxychalcone and other hydroxychalcones inhibited both DOXol and DNRol formation, whereas quercetin and other flavonoids inhibited DNRol formation but failed to inhibit or slightly stimulated DOXol formation. These results identify chalcones as versatile inhibitors of the cytoplasmic reductases that convert anthracyclines to cardiotoxic secondary alcohol metabolites.

BACKGROUND Fibroepithelioma of Pinkus (FeP) is a rare variant of basal cell carcinoma that may clinically mimic a number of benign skin tumors. While the dermoscopic features of basal cell carcinoma have been studied extensively, little is known about the dermoscopic features of FeP. OBSERVATIONS Retrospective evaluation of clinical records and digital clinical dermoscopic images of 10 histopathologically proved FePs (6 nonpigmented and 4 pigmented) was performed. Clinically, no FeP was correctly identified and, in half of all patients, a clinical differential diagnosis of purely benign skin lesions was made. Dermoscopy enabled the correct diagnosis in 9 of 10 FePs, based on the presence of fine arborizing vessels, either alone or associated with dotted vessels, and white streaks (in 100%, 70%, and 90% of lesions, respectively). In the 4 pigmented FePs, a structureless gray-brown area of pigmentation and variable numbers of gray-blue dots were observed, in addition. CONCLUSIONS Dermoscopy is helpful in diagnosing FeP and in differentiating this variant of basal cell carcinoma from other benign skin tumors commonly included in the clinical differential diagnosis. This presumes, however, that dermoscopy is used as a first-line examination for all skin lesions, not only for those that are clinically suspect.Arch Dermatol. 2006;142:1318-1322--

Idebenone is a synthetic analogue of coenzyme Q10 with antioxidant properties. The present study investigated the antioxidant activity of idebenone in the rescue of acoustic trauma. Noise-induced hearing loss was induced by exposing guinea pigs to a continuous pure tone and idebenone was injected intraperitoneally 1 h before noise exposure and once daily for 3 days. Guinea pigs treated with idebenone showed significantly smaller auditory threshold shifts than unprotected control animals. Missing and apoptotic cells were identified with scanning electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Protected animals presented a lesser extent of both apoptotic activation and hair cell loss in the organ of Corti. Our results suggest an antioxidant function of idebenone in protection from noise-induced hearing loss and provide a rationale for exploring therapeutic strategies in humans.

Purpose: To evaluate the efficacy of the addition of tamsulosin to our standard expulsive pharmacologic therapy for the treatment of distal-ureteral stones.Patients and Methods: A series of 96 patients referred to our department for the management of symptomatic distal-ureteral calculi were randomly divided into group 1 (N = 46) who received diclofenac (100 mg/daily) plus aescin (80 mg/daily) and group 2 (N = 50) who received the same therapy plus tamsulosin (0.4 mg/daily) for a maximum of 2 weeks. There were no differences between the groups with respect to age, sex, or stone size. The primary endpoint was the expulsion rate. Expulsion time, need for analgesics, need for hospitalization, and drug side effects were the secondary endpoints.Results: The expulsion rate was significantly higher in group 2 (90%) than in group 1 (58.7%; P= 0.01), and group 2 achieved stone passage in a shorter time (mean 4.4 v 7.5 days, respectively; P= 0.005). Lower analgesic use was found in group 2 (P= 0.003), as well as significantly fewer hospitalizations for recurrent colic (P= 0.01). Both groups experienced few side effects associated with expulsive therapy.Conclusions: A conservative approach should be considered as an option in the management of uncomplicated distal-ureteral stones. Even if the best pharmacologic expulsive regimen remains to be established, the use of the selective α-blocker tamsulosin is recommended in this setting.

Several unrandomized studies from the current literature confirm that in selected patients with localized renal cell carcinoma (RCC), nephron sparing surgery (NSS) shows to be as effective as radical surgery. In this regard, we evaluated the data from patients treated by using such a conservative approach with a long-term follow-up.Materials and Methods. We considered 28 patients (19 M, 9 F; median age 54 years) with unilateral, localized, small (< 4 cm) RCC, submitted to NSS from 1988 to 1994. Only 3 of them (10%) were symptomatic at presentation. Oncological follow-up had been conducted with visits every 4 months for the first two years, every six for another three years and then annually.Results. All the patients were clinical stage T1aN0M0 (UICC TNM 2002). Grading was: 10 G1, 10 G2, 8 G3. Mean tumor diameter was 3.2 cm. After a long-term follow-up (mean 10 years, range 8–14), none present local relapse, disease specific survival is 93% and overall survival is 86%. Based on biochemistry, 82% of the remaining patients still have a normal renal function.Conclusions. Given the excellent long-term results and the recognized benefits of elective NSS, this approach should be preferentially adopted in a selected population of patients with small (<4 cm), unilateral, RCC.

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association.In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine.Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin.All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

Homocysteine is a non-protein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H2O2, enhanced platelet activity and increased production of macrophage-derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine-generated H2O2 has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol-dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol–hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at µmol/L concentrations (10–100 µmol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol–hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H2O2-mediated neutrophil activity is not supported and, probably, the common opinion that views the H2O2 generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered. Copyright © 2000 John Wiley & Sons, Ltd.

Homocysteine is a non‐protein‐forming sulphur amino acid that plays an important role in remethylation and trans‐sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H2O2, enhanced platelet activity and increased production of macrophage‐derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine‐generated H2O2has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol‐dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol–hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at µmol/L concentrations (10–100 µmol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol–hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H2O2‐mediated neutrophil activity is not supported and, probably, the common opinion that views the H2O2generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered. Copyright © 2000 John Wiley & Sons, Ltd.

The anthracycline doxorubicin (DOX) is an exceptionally good antineoplastic agent, but its use is limited by formation of metabolites which induce acute and chronic cardiac toxicities. Whereas the acute toxicity is mild, the chronic toxicity can produce a life-threatening cardiomyopathy. Studies in laboratory animals are of limited value in predicting the structure and reactivity of toxic metabolites in humans; therefore, we used an ethically acceptable system which is suitable for exploring DOX metabolism in human myocardium. The system involves cytosolic fractions from myocardial samples obtained during aorto-coronary bypass grafting. After reconstitution with NADPH and DOX, these fractions generate the alcohol metabolite doxorubicinol (DOXol) as well as DOX deoxyaglycone and DOXol hydroxyaglycone, reflecting reduction of the side chain carbonyl group, reductase-type deglycosidation of the anthracycline, and hydrolase-type deglycosidation followed by carbonyl reduction, respectively. The efficiency of each metabolic route has been evaluated at low and high DOX:protein ratios, reproducing acute, single-dose and chronic, multiple-dose regimens, respectively. Low DOX:protein ratios increase the efficiency of formation of DOX deoxyaglycone and DOXol hydroxyaglycone but decrease that of DOXol. Conversely, high DOX:protein ratios facilitate the formation of DOXol but impair reductase- or hydrolase-type deglycosidation and uncouple hydrolysis from carbonyl reduction, making DOXol accumulate at levels higher than those of DOX deoxyaglycone and DOXol hydroxyaglycone. Structure−activity considerations have suggested that aglycones and DOXol may inflict cardiac damage by inducing oxidative stress or by perturbing iron homeostasis, respectively. Having characterized the influence of DOX:protein ratios on deglycosidation or carbonyl reduction, we propose that the benign acute toxicity should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity should be attributed to alterations of iron homeostasis by DOXol. This picture rationalizes the limited protective efficacy of antioxidants against chronic cardiomyopathy vis-à-vis the better protection offered by iron chelators, and forms the basis for developing analogues which produce less DOXol.

Non enzymatic glycation could be involved in the early impairment of Na+/K+ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ATPase impairment and hydrogen-peroxide formation. No gross structural and confor-mational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements.Our results suggest that during the exposure of brain Na+/K+ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at least in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydro-peroxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20°C. However, on going from 20°C to 37°C, the difference in O₂affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (ΔH) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.

Two-equivalent oxidation of metmyoglobin (MbIII) by hydrogen peroxide (H2O2) yields an oxoferryl moiety (MbIV) plus a protein radical which presumably originates from the conversion of tyrosines to tyrosyl radicals (-MbIV). In the absence of electron donors, MbIII oxidation is followed by (i) heme degradation or (ii) tyrosyl radical-dependent reactions, such as irreversible dimerization or covalent binding of the heme group to the apoprotein. Moreover, the oxidizing equivalents of H2O2-activated MbIII promote the peroxidative decomposition of polyunsaturated fatty acids. In this study, water-soluble short chain coenzyme Q analogs (CoQ1H2 and CoQ2H2) were found to reduce the oxoferryl moiety, preventing heme degradation and regenerating MbIII and, more slowly, MbIIO2. CoQ1H2 and CoQ2H2 were also found to reduce tyrosyl radicals generated by UV irradiation of tyrosine solutions. Accordingly, CoQ1H2 and CoQ2H2 effectively prevented tyrosyl radical-dependent reactions such as the dimerization of sperm whale myoglobin and heme-apoprotein covalent binding in horse heart myoglobin. By competing for the oxidizing equivalents of hypervalent myoglobin, CoQ1H2 and CoQ2H2 also prevented the peroxidation of arachidonic acid. Collectively, these studies suggest that the proposed function of coenzyme Q as a naturally occurring antioxidant might well relate to its ability of reducing H2O2-activated myoglobin. Coenzyme Q should therefore mitigate cardiac or muscular dysfunctions that are caused by an abnormal generation of H2O2.

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of ?SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.

PurposeTo assess cathepsin D (Cat.D) status in the prostate, we analyzed the different Cat.D forms in human prostate tissues using Western immunoblots.Materials and MethodsCell extracts were prepared from prostate tissues (n = 42) obtained from radical prostatectomy, adopting the tissue homogenization method. Expression of the different Cat.D forms was analyzed using Western blots. The catalytic activity of Cat.D was assayed by acid treatment, in which cell extracts were incubated in acidic buffer (pH 3 to 4) at 37C for 1 hour.ResultsPathologically confirmed normal (NML), benign prostatic hyperplasia (BPH) and cancer (CAP) specimens all expressed Cat.D, but as two distinct forms. Both NML and BPH predominantly expressed an inactive procathepsin D (Pro.Cat.D), while CAP notably exhibited an active mature Cat.D. The assessment of Cat.D activity, using PSA (prostate specific antigen) as a physiological substrate, showed that such activity was consistently higher in CAP than in NML/BPH specimens. Further studies revealed that the mode of Cat.D activation in CAP specimens appeared to be primarily due to acid-induced autoproteolysis (self-degradation) of mature Cat.D.ConclusionThis study demonstrates that expression and activity of Cat.D varies among prostate specimens. A greater expression of mature Cat.D with a higher catalytic activity in CAP specimens is the most notable difference from NML/BPH. Therefore, the differential expression/activity activity of Cat.D forms may be a useful indicator for assessing prostate cancer status.

Idebenone [2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone] is a synthetic analogue of coenzyme Q that is currently employed in the treatment of vascular and degenerative diseases of the central nervous system. There is some evidence to suggest that idebenone might function as an antioxidant; however, it has not been demonstrated whether this function pertains to the quinone or hydroquinone form of idebenone. Here we demonstrate that idebenone can scavenge a variety of free radical species, including organic radicals such as 2,2‘-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and diphenylpicrylhydrazyl, peroxyl and tyrosyl radicals, and peroxynitrite. Idebenone can also redox couple with hypervalent species of Mb or Hb, thus preventing lipid peroxidation promoted by these species. Likewise, idebenone inhibits microsomal lipid peroxidation induced by ADP−iron complexes or organic hydroperoxides. In so doing, idebenone prevents the destruction of cytochrome P450, which otherwise would accompany lipid peroxidation. Irrespective of the experimental system under investigation, idebenone functions by virtue of the electron-donating properties of the hydroquinone form. Redox coupling of this hydroquinone with free radicals generates the quinone compound, which per se lacks antioxidant activity. In many experiments, the antioxidant effects of idebenone become appreciable at ~2 μM, which is well in the range of plasma levels attainable in patients given oral doses of this drug. Moreover, comparative experiments have shown that the antioxidant efficiency of idebenone varies from no less than 50% to slightly more than 100% of that of vitamin E or Trolox. We would therefore propose that the neuroprotective effects of idebenone can be attributed, at least in part, to its ability to function as an antioxidant, involving redox cycling between hydroquinone and quinone.

To investigate the effects of brefeldin A (BFA) on the growth of the androgen-independent human prostate cancer PC-3 cells, focusing on cell cycle regulation.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

Moderate concentrations of guanidinium chloride induce both instantaneous and time-dependent modifications of the catalytic and optical properties of intestinal alkaline phosphatase, which undergoes consecutive conformational transitions at about 0.05 M, 0.25 M and 1.0 M denaturant. A paradoxical activation is observed up to 1.0 M-guanidine, with a maximum at 0.25 M- and a mid-point around 0.5 M-guanidine. Difference absorbance and fluorescence spectra imply a change in the state of ionization of the protein residues, with variation in molecular size suggested by light-scattering. Random-coil formation is indicated by a lower fluorescence yield, a more polar environment of the aromatic residues and another separate tryptophan emission. Iodide quenching confirms the alterations of conformation. Deprotonation favours the loss of the intramolecular constraints and the enhancement of the structure disruption by guanidine.