In VitroReconstitution of the Recombinant Photosystem II Light-harvesting Complex CP24 and Its Spectroscopic Characterization

The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a

New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart

Repurposed Biguanide Drugs in Glioblastoma Exert Antiproliferative Effects via the Inhibition of Intracellular Chloride Channel 1 Activity

The lack of in-depth knowledge about the molecular determinants of glioblastoma (GBM) occurrence and progression, combined with few effective and BBB crossing-targeted compounds represents a major challenge for the discovery of novel and efficacious drugs for GBM. Among relevant molecular factors controlling the aggressive behavior of GBM, chloride intracellular channel 1 (CLIC1) represents an emerging prognostic and predictive biomarker, as well as a promising therapeutic target. CLIC1 is a metamorphic protein, co-existing as both soluble cytoplasmic and membrane-associated conformers, with the latter acting as chloride selective ion channel. CLIC1 is involved in several physiological cell functions and its abnormal expression triggers tumor development, favoring tumor cell proliferation, invasion, and metastasis. CLIC1 overexpression is associated with aggressive features of various human solid tumors, including GBM, in which its expression level is correlated with poor prognosis. Moreover, increasing evidence shows that modification of microglia ion channel activity, and CLIC1 in particular, contributes to the development of different neuropathological states and brain tumors. Intriguingly, CLIC1 is constitutively active within cancer stem cells (CSCs), while it seems less relevant for the survival of non-CSC GBM subpopulations and for normal cells. CSCs represent GBM development and progression driving force, being endowed with stem cell-like properties (self-renewal and differentiation), ability to survive therapies, to expand and differentiate, causing tumor recurrence. Downregulation of CLIC1 results in drastic inhibition of GBM CSC proliferation in vitro and in vivo, making the control of the activity this of channel a possible innovative pharmacological target. Recently, drugs belonging to the biguanide class (including metformin) were reported to selectively inhibit CLIC1 activity in CSCs, impairing their viability and invasiveness, but sparing normal stem cells, thus representing potential novel antitumor drugs with a safe toxicological profile. On these premises, we review the most recent insights into the biological role of CLIC1 as a potential selective pharmacological target in GBM. Moreover, we examine old and new drugs able to functionally target CLIC1 activity, discussing the challenges and potential development of CLIC1-targeted therapies

Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration

Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA

High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of "stem-like" cells to render them more susceptible to the killing action of cytotoxic anticancer drugs

Phylogeny and regulation of four lipocalin genes clustered in the chicken genome: evidence of a functional diversification after gene duplication

Producción CientíficaA novel lipocalin gene is here reported that represents the fourth member of a cluster we have identified in the chicken genome. This cluster also includes Chondrogenesis-Associated Lipocalins h and g (CALh, CALg) and Extracellular Fatty Acid Binding Protein (Ex- FABP). The new gene codes for a 22-kDa secreted protein with three cysteine residues and a series of sequence features well conserved in the lipocalin family. All the genes in the cluster are structurally similar presenting comparable exon/intron boundary positions and exon sizes. A phylogenetic analysis indicates the monophyletic grouping of these genes, and their relationship with the lipocalins a-1-microglobulin (A1mg), complement factor 8g chain (C8GC), prostaglandin D synthase (PGDS), and neutrophil-gelatinase-associated lipocalin (NGAL). The new cluster gene appears to be the ortholog of the mammalian C8GC and was thus named Ggal-C8GC. This orthology also suggests that this lipocalin was present in the ancestor common to reptiles and mammals. In addition to other expressing tissues, Ex-FABP, CALh and CALg genes are highly transcribed in chondrocytes at late stages of chondrogenesis during endochondral bone formation and/or upon inflammatory stimulation. Here, we show that they are also transcriptionally induced when chondrocytes are subjected to various biological events as cell quiescence, cell shape transition, and hormonal stimulation. By contrast, Ggal-C8GC transcripts are only barely detectable in chondrocytes, but are more abundant in liver, kidney, brain, heart, skeletal muscle and particularly in skin. Moreover, no expression induction was observed neither during chondrocyte differentiation, nor upon any of the stimulations mentioned above. This indicates that the Ggal-C8GC gene was co-opted for a novel function after the duplication events that gave rise to the cluster. The peculiar coordinated regulation of Ex-FABP, CALh and CALg, and the apparent divergent role of Ggal-C8GC suggest that these gene duplications may have been maintained during evolution by a sub-functionalization mechanism where some common function(s) are shared by several members of the cluster and some other specialized function(s) are unique to other members

14 MeV neutrons for 99Mo/99mTc production: Experiments, simulations and perspectives

Background: the gamma-emitting radionuclide Technetium-99m (99mTc) is still the workhorse of Single Photon Emission Computed Tomography (SPECT) as it is used worldwide for the diagnosis of a variety of phatological conditions.99mTc is obtained from99Mo/99mTc generators as pertechnetate ion, which is the ubiquitous starting material for the preparation of99mTc radiopharmaceuticals.99Mo in such generators is currently produced in nuclear fission reactors as a by-product of235U fission. Here we investigated an alternative route for the production of99Mo by irradiating a natural metallic molybdenum powder using a 14-MeV accelerator-driven neutron source. Methods: after irradiation, an efficient isolation and purification of the final99mTc-pertechnetate was carried out by means of solvent extraction. Monte Carlo simulations allowed reliable predictions of99Mo production rates for a newly designed 14-MeV neutron source (New Sorgentina Fusion Source). Results: in traceable metrological conditions, a level of radionuclidic purity consistent with accepted pharmaceutical quality standards, was achieved. Conclusions: we showed that this source, featuring a nominal neutron emission rate of about 1015s−1, may potentially supply an appreciable fraction of the current99Mo global demand. This study highlights that a robust and viable solution, alternative to nuclear fission reactors, can be accomplished to secure the long-term supply of99Mo

Rolly Protein (ROLP)-Epb4.1/3: A Potential Protein-Protein Interaction Relevant for the Maintenance of Cell Adhesion

We recently described Rolly Protein (ROLP), a small protein synthesized by substrate-adherent cells in a broad range of tissues. In a first set of experiments performed taking advantage of bone forming tibial cartilage as an experimental model we showed that ROLP transcription is associated to cells in an active proliferation state, whereas its downregulation is observed when cell proliferation decreases. Taking advantage of siRNA technology we also documented the expression modulation of some apoptosis-related genes in ROLP-silenced cells. In this work we search for the possible molecular interactors of ROLP by using both the antibody array approach as well as the co-immunoprecipitation approach. Results suggest the occurrence of an interaction of ROLP with Erythrocyte membrane Protein Band 4.1/3 (Epb4.1/3), an oncosuppressor downregulated in tumor development and in metastatic tissues; in addition we report experimental results that keep in line also with a potential interaction of ROLP with other PDZ-containing proteins. We also present experimental evidences supporting a role played by ROLP in cell adhesion thus supporting the existence of a biologically relevant link between ROLP and Epb4.1/3. We here suggest that ROLP might exert its biological role cooperating with Epb4.1/3, a protein that is involved in biological pathways that are often inhibited in tumor metastasis. Given the role of Epb4.1/3 in contrasting cancerogenesis we think that its cooperation with ROLP might be relevant in cancer studies and deserves further investigation

Pharmacological activation of autophagy favors the clearing of intracellular aggregates of misfolded prion protein peptide to prevent neuronal death

According to the "gain-of-toxicity mechanism", neuronal loss during cerebral proteinopathies is caused by accumulation of aggregation-prone conformers of misfolded cellular proteins, although it is still debated which aggregation state actually corresponds to the neurotoxic entity. Autophagy, originally described as a variant of programmed cell death, is now emerging as a crucial mechanism for cell survival in response to a variety of cell stressors, including nutrient deprivation, damage of cytoplasmic organelles, or accumulation of misfolded proteins. Impairment of autophagic flux in neurons often associates with neurodegeneration during cerebral amyloidosis, suggesting a role in clearing neurons from aggregation-prone misfolded proteins. Thus, autophagy may represent a target for innovative therapies. In this work, we show that alterations of autophagy progression occur in neurons following in vitro exposure to the amyloidogenic and neurotoxic prion protein-derived peptide PrP90-231. We report that the increase of autophagic flux represents a strategy adopted by neurons to survive the intracellular accumulation of misfolded PrP90-231. In particular, PrP90-231 internalization in A1 murine mesencephalic neurons occurs in acidic structures, showing electron microscopy hallmarks of autophagosomes and autophagolysosomes. However, these structures do not undergo resolution and accumulate in cytosol, suggesting that, in the presence of PrP90-231, autophagy is activated but its progression is impaired; the inability to clear PrP90-231 via autophagy induces cytotoxicity, causing impairment of lysosomal integrity and cytosolic diffusion of hydrolytic enzymes. Conversely, the induction of autophagy by pharmacological  blockade of mTOR kinase or trophic factor deprivation restored autophagy resolution, reducing intracellular PrP90-231 accumulation and neuronal death. Taken together, these data indicate that PrP90-231 internalization induces an autophagic defensive response in A1 neurons, although incomplete and insufficient to grant survival; the pharmacological enhancement of this process exerts neuroprotection favoring the clearing of the internalized peptide and could represents a promising neuroprotective tool for neurodegenerative proteinopathies

Design, synthesis, biophysical and biological studies of trisubstitutednaphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)3 binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UVmelting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment