Mode of membrane insertion and sequence of a 32-amino acid peptide stretch of the penicillin-binding protein 4 of Enterococcus hirae

Analysis of water-soluble derivatives of the Enterococcus hirae 75-kDa membrane-bound penicillin-binding protein 4 (PBP4) has yielded the amino acid sequence of a 32-amino acid polypeptide stretch. This peptide is similar to peptide segments known to occur in the N-terminal domain of high-Mr PBPs of class B. The E. hirae PBP4 probably belongs to the same class. It is anchored in the membrane at the N-terminus of the polypeptide chain.     

Angiotensin II: Immunohistochemical Study in Sardinian Pterygium

The Angiotensin II (Ang II) is the principal effector peptide of the RAS system. It has a pleiotropic effect and, beside its physiological role, it has the property to stimulate angiogenesis and activate multiple signalling pathways related to cell proliferation. The purpose of the study was to determinate the Ang II expression and localization in Sardinian pterygium and normal conjunctiva by immunohistochemistry, and its possible involvement in the development and progression of the disease. Twenty-three pterygiums and eleven normal conjunctiva specimens obtained from Sardinian patients, were processed for paraffin embedding and assessed for the immunohistochemi-cal revelation of Ang II. Significant Ang II expression was identified in pterygium and conjunctiva. Particularly, thirteen pterygium specimens (n=13) displayed exclusively moderate to strong nuclear staining; some specimens (n=5) showed exclusively a moderate cytoplasmic immunoreactivity, and few specimens (n=2) displayed moderate to strong immunoreactivity in both cytoplasm and nucleus. Only 3 specimens were negative. Statistical significance difference in respect of nuclear and cytoplasmic localization was observed between normal conjunctiva and pterygium (P=0.020). The results showed a predominant intranuclear localization of Ang II in pterygium epithelial cells, in spite of conjunctiva that mainly showed cytoplasmic localization. These findings suggest a possible role for Ang II in the development and/or progression of pterygium mediated by the activation of local RAS system.Key words: Renin-angiotensin system (RAS), angiotensin II (Ang II), intracellular, immunohistochemistry     

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

Ozone effects on carbon metabolism in sensitive and insensitive Phaseolus cultivars

An ozone treatment of 165 nL L^?1 for 3 h evoked differential responses in the primary leaves of two bean cultivars of the common bean (Phaseolus vulgaris L.). While cv ‘Cannellino’ showed visible symptoms of injury within 24 h of exposure, no visible symptoms at all were evident in the cv ‘Top Crop’. In primary leaves of the sensitive cultivar Cannellino, we observed an increase in carbon breakdown (an increase in PFK and Fumarase) and a reduction in CO₂ photoassimilation, linked also to the diminished synthesis of sucrose (a decrease in SPS activity) and to the stimulation of the degradation of this sugar (an increase in SuSy and Invertase activities). A strong stimulation of PEPcase activity indicates both an increased synthesis of OAA and an enhanced replenishment of the tricarboxylic acid cycle. Finally, in Cannellino leaves the activity of NADP-malic enzyme increased indicating a stimulation of enzymes delivering NADPH. The findings of this research suggest that the visible symptoms in Cannellino represent an active response that this cultivar initiates to cope with excess oxidative load. The pattern in Top Crop was different. This cultivar did not show visible symptoms of injury, nor any responsive changes at the physiological and biochemical levels. Oxidative pathways are partially enhanced, e.g., increases in Invertase, PFK and IDH. There were also increases in some enzyme linked to the production of cytosolic NADPH as G6PD, probably caused by the slight increase in activity of the enzymes SKDH and PAL involved in synthesis of phenolic compounds. However, the absence of visible injury in Top Crop leaves is confirmation that, in this cultivar, the need to produce carbon skeleton and NADPH used in detoxification and repair process is lower.     

Ret, Abl1 (cAbl) and Trp53 gene fragmentations in comet-FISH assay act as in vivo biomarkers of radiation exposure in C57BL/6 and CBA/J mice

The International Commission on Radiation Protection (ICRP) has lowered the dose limits for workers and for the general public exposed to ionizing radiation. Consequently, a reliable dosimetric method for monitoring possible radiation-induced damage is of great importance in radioprotection. The counting of dicentric chromosomal aberrations and of micronuclei in peripheral blood lymphocytes is unreliable when it is applied to in vivo biopsies and for low-dose exposures. Single-cell gel electrophoresis (SCGE or comet assay), although sensitive and rapid, shows high variability when applied in vivo, probably due to prompt repair of the DNA breaks and confounding environmental factors. In this paper, we describe specific in situ hybridization of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on SCGE slides (comet-FISH assay) in peripheral blood cells from C57BL/6 and CBA/J mice as an indicator of radiation-induced DNA damage. The results obtained from four mice for each experimental point (0, 1, 2 and 4 Gy of X rays) discriminated in a statistically significant way the effects of all doses when fragmentations were analyzed for the Ret, Ab1 and Trp53 genes. SCGE alone, when applied to the same specimens, produced no significant results because of interindividual and experimental variability.     

New pyridazinone derivatives with vasorelaxant and platelet antiaggregatory activities

New 6-substituted and 2,6-disubstituted pyridazinone derivatives were obtained starting from easily accessible alkyl furans by using oxidation with singlet oxygen to give 4-methoxy or 4-hydroxybutenolides, key intermediates of this synthetic strategy. The new pyridazinone derivatives have been studied as vasorelaxant and antiplatelet agents. Analysis of biological data revealed the silyl ethers (4a–i) and N,O-dibenzyl derivatives (6g–i) as the most active compounds.     

Ligand-based virtual screening and ADME-tox guided approach to identify triazolo-quinoxalines as folate cycle inhibitors

In the process of drug discovery the lead-identification phase may be critical due to the likely poor safety profile of the candidates, causing the delay or even the abandonment of a certain project. Nowadays, combining molecular modeling and in vivo cellular evaluation can help to identify compounds with an enhanced safety profile. Previously, two quinoxalines have been identified as inhibitors of the folate-dependent proteins belonging to the thymidylate synthase cycle. Unfortunately, cytotoxic activity against a panel of cisplatin(cDDP)-sensitive ovarian carcinoma cell lines and their resistant counterparts was coupled with toxicity to non-tumorigenic Vero cells. Here we describe the application of a ligand-based virtual screening, and several [1,2,4]triazolo[4,3-a]quinoxalines were optimized to improve their ADME-tox profile. The resulting 4-(trifluoromethyl)-1-p-tolyl-[1,2,4]triazolo[4,3-a]quinoxaline (24), which interferes intracellularly with DHFR and TS reducing the protein levels like 5-FU, but without inducing TS ternary complex formation, was 2-times less toxic in vitro than cisplatin and 5-FU.     

A 450 million years long latitudinal gradient in age‐dependent extinction

Leigh Van Valen famously stated that under constant conditions extinction probability is independent of species age. To test this 'law of constant extinction', we developed a new method using deep learning to infer age‐dependent extinction and analysed 450 myr of marine life across 21 invertebrate clades. We show that extinction rate significantly decreases with age in > 90% of the cases, indicating that most species died out soon after their appearance while those which survived experienced ever decreasing extinction risk. This age‐dependent extinction pattern is stronger towards the Equator and holds true when the potential effects of mass extinctions and taxonomic inflation are accounted for. These results suggest that the effect of biological interactions on age‐dependent extinction rate is more intense towards the tropics. We propose that the latitudinal diversity gradient and selection at the species level account for this exceptional, yet little recognised, macroevolutionary and macroecological pattern.     

Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.