Anti-oxidative and photo-protective effects of coumarins isolated fromFraxinus chinensis

Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may cause serious injury to skin cell membranes, DNA and functional proteins. In addition, these agents stimulate the expressions of matrix metalloproteinases (MMPs), which can degrade most components of the extracellular matrix (ECM), including collagen. In order to develop new anti-photoaging agents, five major components from the extract of Fraxinus chinensis extract (FCE) were identified. Two of the major components of FCE were found to be esculin (11.2%) and esculetin (1.9%). FCE (IC50: 50.0 microg/mL 1, 1-diphenyl-2-picrylhydrazyl (DPPH); 19.8 microg/mL, superoxide anion radical) and esculetin (IC50: 2.1 microg/mL DPPH; 0.6 microg/mL, superoxide anion radical) showed strong antioxidative activities. Of the compounds tested, esculetin showed the strongest scavenging activity against DPPH radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. The intracellular ROS scavenging activity showed that oxidation of 5-(6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was effectively inhibited by esculetin, with potent free radical scavenging activity was also shown in UVB-irradiated human dermal fibroblasts (HDFs). Moreover, treatment of UVA-irradiated HDFs with esculetin resulted in dose-dependent decreases in the expression levels of MMP-1 mRNA and protein. From these results, FCE and one of its components, esculetin, were predicted to be potentially useful as ingredients in cosmetics for protecting against photoaging.     

Metabolism of sanguinarine: the facts and the myths

Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid, exhibits antimicrobial and anti-inflammatory activities and for this reason it is used in dental hygiene products and feed additives. Its metabolism and disposition is the subject of constant scientific discourse. In this paper we summarize current knowledge on sanguinarine metabolism. We show in particular that: (i) Sanguinarine is not transformed to 3,4-benzacridine and that the literature reporting this compound as a metabolite of sanguinarine is based on artifacts and misinterpretations that in course of time have created a dogma; (ii) Sanguinarine is converted to dihydrosanguinarine in vivo, the conversion being tentatively a detoxication pathway; (iii) Aryl hydrocarbon receptor metabolic signaling pathways modulate sanguinarine biological activity.     

Highly Penicillin-Resistant Multidrug-Resistant Pneumococcus-Like Strains Colonizing Children in Oeiras,Portugal: Genomic Characteristics and Implications for Surveillance

While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as “streptococci of the mitis group” that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006.Streptococcus pneumoniae is a bacterial pathogen that frequently colonizes the nasopharynx of humans, particularly young children of preschool age. Colonization is mostly asymptomatic and only rarely results in disease (3). However, when disease does occur, it may range from a mild infection such as otitis media to severe septicemia or meningitis. Globally, the morbidity and mortality associated with pneumococcal infections are extremely high. A recent report from the WHO estimated that 0.7 to 1.0 million deaths occur annually among children <5 years of age as a result of pneumococcal infections (50).Four phenotypic characteristics are classically used in the diagnostic laboratory for the presumptive identification of S. pneumoniae: colony morphology (colonies with a depression in the center showing alpha-hemolysis on sheep blood agar), optochin susceptibility, deoxycholate (DOC) solubility (commonly referred to as bile solubility), and a positive reaction with antipneumococcal polysaccharide capsule antibodies (20). In particular, optochin susceptibility and deoxycholate solubility have been associated with high sensitivity and specificity (between 98% and 100%). However, a number of studies have reported on sporadic optochin-resistant S. pneumoniae isolates (28, 34) and rare deoxycholate-insoluble strains (32).On the other hand, some nonpneumococcal oral streptococci (such as Streptococcus mitis) may have a colony morphology similar to that of pneumococci but are classically optochin resistant, bile insoluble, and do not react with antipneumococcal polysaccharide capsule antibodies (25). Still, optochin-susceptible nonpneumococcal isolates have been described occasionally (20), as well as isolates with positive cross-reactions with antipneumococcal polysaccharide capsule antibodies (11). Recently, a new species—Streptococcus pseudopneumoniae—was described (1). S. pseudopneumoniae isolates were found to be resistant to optochin when incubated in an atmosphere enriched in CO₂ but were optochin susceptible when incubated in ambient atmosphere. As a result, S. pneumoniae isolates may be difficult to distinguish from closely related species such as S. pseudopneumoniae and S. mitis.Since the biochemical tests commonly used are not always sufficient to distinguish S. pneumoniae from other closely related upper respiratory streptococci, molecular approaches based on amplification of ubiquitous pneumococcal genes, such as pneumolysin (ply) and autolysin (lytA), have been proposed (27). However, homologues of lytA and ply genes have been detected in strains of closely related streptococcal species (15, 29, 37, 49). Recently, detection of piaA (which encodes a lipoprotein component of two iron ABC transporters) has been proposed as a diagnostic tool for pneumococci as it was suggested to be specific for this species (47). Some authors also described 16S rRNA and sodA as good targets for identification of S. pneumoniae although sodA does not distinguish S. pneumoniae from S. pseudopneumoniae (1, 12, 21). The construction of phylogenetic trees from the concatenated sequences of the genes used for multilocus sequence typing (MLST)—an approach commonly termed multilocus sequence analysis (MLSA)—has also been proposed as a good alternative molecular technique to differentiate pneumococci from other closely related streptococci (9, 18).In recent years, we have been studying atypical pneumococci recovered from colonization samples collected from children attending day care centers (DCCs) in Portugal. We first described a collection of over 200 nonserotypeable pneumococci which were mostly multidrug resistant and displayed low-level resistance to penicillin. All isolates were found to be true pneumococci that lacked a capsular operon. The isolates were genetically diverse although close to half belonged to a single lineage (39). Overall, these isolates were relatively abundant in asymptomatic carriers. The second group of atypical pneumococci that we described consisted of isolates resistant to optochin. Again, all strains were found to be true pneumococci and genetically diverse, and, in this case, most expressed a pneumococcal capsular type (30).In the current study, we describe a third set of presumptively identified atypical pneumococcal strains. These isolates were all obtained in 2006 during a cross-sectional study designed to describe the impact of the seven-valent pneumococcal conjugate vaccine in colonization. Strikingly, 5% (22 of 441) of the presumptively identified pneumococcal isolates were found to have penicillin MICs ranging from 2 to 6 mg/liter, a value that between 2001 and 2003 never exceeded 1.7% and was 0.7% in 2003. Of note, 15 of these isolates, although resistant to optochin, were bile soluble and, thus, according to routinely accepted criteria, were considered to be bona fide pneumococci. Since such high MICs of penicillin are extremely rare among this population, we initiated a detailed characterization of these isolates consisting of classical strategies for pneumococcal identification, MLSA, and comparative genomic hybridization (CGH). Ten other optochin-resistant, bile-soluble isolates with penicillin MICs ranging from 0.064 to 0.75 mg/liter were also identified and further characterized.We report here that these isolates are not true pneumococci but have phenotypic properties and genomic determinants that are frequently associated with S. pneumoniae, challenging their correct identification by several currently accepted assays.     

Germline mutations of BRCA1 and BRCA2 genes in Turkish breast, ovarian, and prostate cancer patients

Distribution and prevalence of germline mutations in BRCA1 and BRCA2 differ among different populations. For the Turkish population, several studies have addressed high-risk breast cancer and ovarian cancer (BC–OC) patients. In most studies, both genes were analyzed in part, and a quite heterogeneous mutation spectrum was observed. For high-risk Turkish prostate cancer (PCa) patients, however, there are no data available about mutations of germline BRCA genes. To accurately determine the contribution of germline mutations in BRCA1 and BRCA2 in Turkish BC, OC, and PCa high-risk patients, 106 high-risk BC–OC patients, 50 high-risk PCa patients, and 50 control subjects were recruited. The study represents the only full screening, to date, of a large series of Turkish high-risk BC–OC patients and the only study in Turkish high-risk PCa patients. Mutation screenings were performed on coding exons of both genes with either denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, or with both techniques. Three deleterious mutations in BRCA1 and three deleterious mutations in BRCA2 were detected in different BC–OC patients, and one truncating mutation was detected in a high-risk PCa patient. In addition, 28 different unclassified and mostly novel variants were detected in both genes, as well as several silent polymorphisms. These findings reflect the genetic heterogeneity of the Turkish population and are relevant to genetic counseling and clinical management.     

Increased complement classical and mannan-binding lectin pathway activities in schizophrenia

Schizophrenia is a severe mental disorder, with worldwide prevalence of 1-1.5%. Immunological research in schizophrenia indicates that infectious or autoimmune processes might play a role in the etiopathogenesis. The complement system is a major mediator of innate immune defence against infection and contributes to many functions of the immune system including inflammation, opsonization and cell lysis. Mannan-binding lectin (MBL) activates the complement system via the lectin pathway. Inherited MBL deficiency, common in most human populations, predisposes to infectious and autoimmune diseases. We measured total complement activity (CH50), C4 activity (C4 CH50), MBL level and the activities of MBL-associated serine proteases, MASP-1 and MASP-2 in sera of 45 schizophrenic patients and in 62 healthy volunteers. We found that schizophrenic patients and healthy volunteers have statistically similar MBL levels and MASP-1 activity. However, MBL-bound MASP-2 activity and therefore MBL and MASP-2-mediated complement activation capacity is increased in schizophrenic patients compared with healthy volunteers (P<0.01). The increase was accompanied by increased CH50 (P<0.02) and C4 CH50 (P<0.02). Our results support the idea that complement system alterations may be involved in schizophrenia.     

Analysis of the monocyte chemoattractant protein 1 -2518 promoter polymorphism in Korean patients with alopecia areata

Monocyte chemoattractant protein-1 (MCP-1) levels are increased in scalp lesions of patients with alopecia areata (AA), suggesting a role in the development of AA. Recently, a biallelic A/G polymorphism in the MCP-1 promoter at position -2518 has been found, influencing the level of MCP-1 expression in response to an inflammatory stimulus. We investigated whether the presence of these polymorphisms were associated with AA in Korean population. 145 Korean patients with AA, 246 healthy subjects without clinical evidence of AA were screened for genotype with a PCR-based assay. In the AA patients the frequency of the A and G alleles was 40.3 and 59.7%, respectively and the distribution of the A/A, A/G and G/G genotypes was 19.3, 42.1 and 38.6%, respectively. Amongst the controls the frequency of the A and G alleles was 39.8 and 60.2%, and the distribution of the A/A, A/G, G/G genotypes in the same group was 17.5, 44.7 and 37.8%, respectively. There was no significant difference in the allele frequencies and genotype distributions between the patients and the controls (p=0.889, p=0.848, respectively). Our data indicates that no association exists between the -2518A/G polymorphism of the MCP-1 gene and susceptibility to alopecia areata.     

Role of catecholaminergic neurones of the caudal ventrolateral medulla in cardiovascular responses induced by acute changes in circulating volume in rats

Several findings suggest that catecholaminergic neurones in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis and cardiovascular regulation. The present study sought to determine the effects of lesions of these neurones on the cardiovascular responses induced by changes in circulating volume. All experiments were performed in male Wistar rats (320-360 g). Medullary catecholaminergic neurones were lesioned by microinjection of anti-dopamine beta-hydroxylase-saporin (6.3 ng in 60 nl; SAP rats, n = 14) into the CVLM, whereas sham rats received microinjections of free saporin (1.3 ng in 60 nl, n = 15). Two weeks later, rats were anaesthetized (urethane, 1.2 g kg(-1), i.v.), instrumented for measurement of mean arterial pressure (MAP), renal blood flow (RBF) and renal vascular conductance (RVC), and infused with hypertonic saline (HS; 3 m NaCl, 0.18 ml (100 g body weight)(-1), i.v.) or an isotonic solution (volume expansion, VE; 4% Ficoll, 1% of body weight, i.v.). In sham rats, HS induced sustained increases in RBF and RVC (155 +/- 7 and 145 +/- 6% of baseline, at 20 min after HS). In SAP rats, RBF responses to HS were blunted (125 +/- 6%) and RVC increases were abolished (108 +/- 5%) 20 min after HS. Isotonic solution increased RBF and RVC in sham rats (149 +/- 10 and 145 +/- 12% of baseline, respectively, at 20 min). These responses were reduced in SAP rats (131 +/- 6 and 126 +/- 5%, respectively, at 20 min). Pressor responses to HS were larger in SAP rats than in sham rats (17 +/- 5 versus 9 +/- 2 mmHg, at 20 min), whereas during VE these responses were similar in both groups (6 +/- 3 versus 4 +/- 6 mmHg, at 20 min). Immunohistochemical analysis indicates that microinjections of anti-DbetaH-saporin produced extensive destruction within the A1/C1 cell groups in the CVLM. These results suggest that catecholaminergic neurones mediate the cardiovascular responses to VE or increases in plasma sodium levels.     

The changes of skin temperature on hands and feet during and after T3 sympathicotomy for palmar hyperhidrosis

Unilateral thoracic sympathectomy in patients with palmar hyperhidrosis causes a skin temperature drop in the contralateral hand. A cross-inhibitory effect by the post-ganglionic neurons innervating hands is postulated as a mechanism of contralateral vasoconstriction. The purpose of our study was to evaluate whether this cross-inhibitory effect also occurs in the feet. Twenty patients scheduled for thoracoscopic sympathicotomy due to palmar hyperhidosis were studied. Right T3 sympathicotomy was performed first, followed by left T3 sympathicotomy. The thenar skin temperatures of both hands and feet were continuously monitored using a thermometer and recorded before induction of anesthesia, during the operation, 4 hr after and 1 week later. Following right T3 sympathicotomy, the skin temperature of the ipsilateral hand gradually increased, however the skin temperature of the contralateral hand gradually decreased. Immediately after bilateral sympathicotomy, the skin temperature differences between hands and feet increased, but these differences decreased 1 week later. Our results show that cross-inhibitory control may exist in feet as well as in the contralateral hand. Thus, the release of cross-inhibitory control following T3 sympathicotomy results in vasoconstriction and decrease of skin temperature on the contralateral hand and feet. One week later, however, the temperature balance on hands and feet recovers.     

Collagen birefringence in skin repair in response to red polarized-laser therapy

We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low-intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L(parallel) is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L(perpendicular) is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L(parallel) and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized.