Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

Identification of paraoxonase 3 gene (PON3) missense mutations in a population of southern Italy

PON gene family includes at least three members termed PON1, PON2 and PON3, and it is mapped on human chromosome 7q21-q22. PON1 and PON3 gene products are constituents of high density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. PON1 and PON2 genes have missense polymorphisms, but, to date, no missense variants are reported in PON3 gene. In this work we explored the existence of genetic variants within the PON3 coding sequences. Five point mutations were identified by direct sequencing of genomic DNA derived from 250 randomly selected DNA samples of 1143 blood donors living in southern Italy. Three were silent mutations, while two were missense mutations that give rise to amino acid substitutions at positions 311 (S>T) and 324 (G>D). The missense variations in the DNA of the 1143 samples had frequencies of 0.22% (5 out of 2286 alleles) for the S311T mutation, and 0.57% (13 out of 2286 alleles) for the G324D mutation. The effect of these variants on the metabolic activity of paraoxonase 3 remains to be further evaluated.     

Diterpenoid resin acids of Daemonorops draco

Chromatography of a cyclohexane extract of commercial “dragon's blood” resin yielded a fraction containing pimaric, isopimaric, dehydroabietic and abietic acids. A fifth component of the mixture was tentatively identified as sandaracopimaric acid.     

Candidate Luminal B Breast Cancer Genes Identified by Genome,Gene Expression and DNA Methylation Profiling

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.     

Is That Me or My Twin? Lack of Self-Face Recognition Advantage in Identical Twins

Despite the increasing interest in twin studies and the stunning amount of research on face recognition, the ability of adult identical twins to discriminate their own faces from those of their co-twins has been scarcely investigated. One’s own face is the most distinctive feature of the bodily self, and people typically show a clear advantage in recognizing their own face even more than other very familiar identities. Given the very high level of resemblance of their faces, monozygotic twins represent a unique model for exploring self-face processing. Herein we examined the ability of monozygotic twins to distinguish their own face from the face of their co-twin and of a highly familiar individual. Results show that twins equally recognize their own face and their twin’s face. This lack of self-face advantage was negatively predicted by how much they felt physically similar to their co-twin and by their anxious or avoidant attachment style. We speculate that in monozygotic twins, the visual representation of the self-face overlaps with that of the co-twin. Thus, to distinguish the self from the co-twin, monozygotic twins have to rely much more than control participants on the multisensory integration processes upon which the sense of bodily self is based. Moreover, in keeping with the notion that attachment style influences perception of self and significant others, we propose that the observed self/co-twin confusion may depend upon insecure attachment.     

Ceftriaxone increases glutamate uptake and reduces striatal tyrosine hydroxylase loss in 6-OHDA Parkinson's model

Excess glutamatergic neurotransmission may contribute to excitotoxic loss of nigrostriatal neurons in Parkinson's disease (PD). Here, we determined if increasing glutamate uptake could reduce the extent of tyrosine hydroxylase (TH) loss in PD progression. The beta-lactam antibiotic, ceftriaxone, increases the expression of glutamate transporter 1 (GLT-1), a glutamate transporter that plays a major role in glutamate clearance in central nervous system and may attenuate adverse behavioral or neurobiological function in other neurodegenerative disease models. In association with >80 % TH loss, we observed a significant decrease in glutamate uptake in the established 6-hydroxydopamine (6-OHDA) PD model. Ceftriaxone (200 mg/kg, i.p.) increased striatal glutamate uptake with >5 consecutive days of injection in nonlesioned rats and lasted out to 14 days postinjection, a time beyond that required for 6-OHDA to produce >70 % TH loss (～9 days). When ceftriaxone was given at the time of 6-OHDA, TH loss was ～57 % compared to ～85 % in temporally matched vehicle-injected controls and amphetamine-induced rotation was reduced about 2-fold. This attenuation of TH loss was associated with increased glutamate uptake, increased GLT-1 expression, and reduced Serine 19 TH phosphorylation, a calcium-dependent target specific for nigrostriatal neurons. These results reveal that glutamate uptake can be targeted in a PD model, decrease the rate of TH loss in a calcium-dependent manner, and attenuate locomotor behavior associated with 6-OHDA lesion. Given that detection of reliable PD markers will eventually be employed in susceptible populations, our results give credence to the possibility that increasing glutamate uptake may prolong the time period before locomotor impairment occurs.     

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.     

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

Background and Aims

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids.

Methods

Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network.

Key Results

Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine.

Conclusions

Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.     

Carbon catabolite regulation of rebeccamycin production inSaccharothrix aerocolonigenes

Summary A new antitumor antibiotic named rebeccamycin was isolated from fermentations of an actinomycete,Saccharothrix aerocolonigenes. A defined medium was developed to study the regulation of synthesis of rebeccamycin byS. aerocolonigenes. In glucose medium formation of rebeccamycin was detected only after glucose was depleted. Examination of eleven different carbon sources revealed that carbon catabolite regulation is a major control mechanism for rebeccamycin production.