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Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. The BM niche is a highly vascularized tissue and endothelial cells (ECs) support and regulate blood cell formation from hematopoietic stem cells (HSCs). While in vivo studies are limited, ECs when cultured in vitro at low O2 (<5%), fail to support functional HSC maintenance due to oxidative environment. Therefore, changes in EC redox status induced by antioxidant molecules may lead to alterations in the cellular response to hypoxia likely favoring HSC self‐renewal. To evaluate the impact of redox regulation, HUVEC, exposed for 1, 6, and 24 h to 3% O2 were treated with N‐(N‐acetyl‐l‐cysteinyl)‐S‐acetylcysteamine (I‐152). Metabolomic analyses revealed that I‐152 increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system and the redox couples NAD(P)+/NAD(P)H. mRNA analysis showed a lowered gene expression of HIF‐1α and VEGF following I‐152 treatment whereas TRX1 and 2 were stimulated. Accordingly, the proteomic study revealed the redox‐dependent upregulation of thioredoxin and peroxiredoxins that, together with the glutathione system, are the main regulators of intracellular ROS. Indeed, a time‐dependent ROS production under hypoxia and a quenching effect of the molecule were evidenced. At the secretome level, the molecule downregulated IL‐6, MCP‐1, and PDGF‐bb. These results suggest that redox modulation by I‐152 reduces oxidative stress and ROS level in hypoxic ECs and may be a strategy to fine‐tune the environment of an in vitro BM niche able to support functional HSC maintenance. [ABSTRACT FROM AUTHOR]

Ataxia‐Telangiectasia (A‐T) is a very rare autosomal recessive multisystemic disorder which to date is still uncurable. The use of glucocorticoid analogs, such as dexamethasone (dex), can improve neurological symptoms in patients, but the molecular mechanism of action of these analogs remains unclear. Here, we report the effects of dex in regulating the interaction between Lamin A/C and HDAC2 in WT and A‐T cells. Upon administration of dex to A‐T cells, we first observed that the accumulation of HDAC2 on the CDKN1A promoter did not exert a repressive role on p21cip1/waf1 expression, and second, we established that HDAC2 accumulation was not dependent on Lamin A/C. Both of these results are contrary to previous reported outcomes in other cellular models. Furthermore, large amounts of LAP2α and FoxO3a were found to occupy the CDKN1A promoter with matched p21cip1/waf1 overexpression. Hence, in A‐T cells p21 could be activated as a result of a dex‐induced rearrangement of a multicomponent complex, composed of Lamin A/C, HDAC2, LAP2α, pRb, E2F1, and FoxO3a, at the CDKN1A gene promoter. [ABSTRACT FROM AUTHOR]

Background: Hypohidrotic ectodermal dysplasia (HED) is the most common form of ectodermal dysplasia and is mainly associated with mutations in the EDA, EDAR, and EDARADD responsible for the development of ectodermal‐derived structures. HED displays different modes of inheritance according to the gene that is involved, with X‐linked EDA‐related HED being the most frequent form of the disease. Methods: Two families with tooth agenesis and manifestations of HED underwent clinical examination and EDA, EDAR, and EDARADD genetic analysis. The impact of the novel variant on the protein was evaluated through bioinformatics tools, whereas molecular modeling was used to predict the effect on the protein structure. Results: A novel missense variant was identified in the EDAR (c.287T>C, p.Phe96Ser) of a female child proband and her mother, accounting for autosomal dominant HED. The genetic variant c.866G>A (p.Arg289His) in EDA, which has been previously described, was observed in the male proband of another family confirming its role in X‐linked HED. The inheritance model of the missense mutation showed a different relationship with X‐linked HED and non‐syndromic tooth agenesis. Conclusion: Our findings provide evidence of variable expression of HED in heterozygous females, which should be considered for genetic counseling, and different modes of inheritance related to tooth development. [ABSTRACT FROM AUTHOR]

Oxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn‐dependent hexameric enzyme that converts oxalate to carbon dioxide and formate. OxDC has greatly attracted the interest of the scientific community, mainly due to its biotechnological and medical applications in particular for the treatment of hyperoxaluria, a group of pathologic conditions caused by oxalate accumulation. The enzyme has an acidic optimum pH, but most of its applications involve processes occurring at neutral pH. Nevertheless, a detailed biochemical characterization of the enzyme at neutral pH is lacking. Here, we compared the structural–functional properties at acidic and neutral pH of wild‐type OxDC and of a mutant form, called OxDC‐DSSN, bearing four amino acid substitutions in the lid (Ser161‐to‐Asp, Glu162‐to‐Ser, Asn163‐toSer, and Ser164‐to‐Asn) that improve the oxalate oxidase activity and almost abolish the decarboxylase activity. We found that both enzymatic forms do not undergo major structural changes as a function of pH, although OxDC‐DSSN displays an increased tendency to aggregation, which is counteracted by the presence of an active‐site ligand. Notably, OxDC and OxDC‐DSSN at pH 7.2 retain 7 and 15% activity, respectively, which is sufficient to degrade oxalate in a cellular model of primary hyperoxaluria type I, a rare inherited disease caused by excessive endogenous oxalate production. The significance of the data in the light of the possible use of OxDC as biological drug is discussed. © 2019 IUBMB Life, 1–11, 2019 [ABSTRACT FROM AUTHOR]

During aging, glutathione (GSH) content declines and the immune system undergoes a deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is associated with GSH depletion, could weaken the host defenses against viral infections. We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the effect of the administration of the N‐butanoyl GSH derivative (GSH‐C4) on the response of aged mice infected with influenza A PR8/H1N1 virus was studied through the determination of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production, and Th1/Th2 cytokine profile. Old mice had lower GSH than young mice in organs. Also the gene expression of endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of proteins, that is, Nrf2 and PDI, was reduced. Following infection, GSH content remained low and neither infection nor GSH‐C4 treatment affected Nrf2 expression. In contrast, PDI expression was upregulated during infection and appeared counterbalanced by GSH‐C4. Moreover, the treatment with GSH‐C4 increased GSH content in organs, reduced viral replication and induced a predominant Th1 response. In conclusion, GSH‐C4 treatment could be used in the elderly to contrast influenza virus infection by inducing immune response, in particular the Th1 profile. [ABSTRACT FROM AUTHOR]

The polyubiquitin gene ubiquitin C (UBC) is considered a stress protective gene and is upregulated under various stressful conditions, which is probably a consequence of an increased demand for ubiquitin in order to remove toxic misfolded proteins. We previously identified heat shock elements (HSEs) within the UBC promoter, which are responsible for heat shock factor (HSF)1‐driven induction of the UBC gene and are activated by proteotoxic stress. Here, we determined the molecular players driving the UBC gene transcriptional response to arsenite treatment, mainly addressing the role of the nuclear factor‐erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant pathway. Exposure of HeLa cells to arsenite caused a time‐dependent increase of UBC mRNA, while cell viability and proteasome activity were not affected. Nuclear accumulation of HSF1 and Nrf2 transcription factors was detected upon both arsenite and MG132 treatment, while HSF2 nuclear levels increased in MG132‐treated cells. Notably, siRNA‐mediated knockdown of Nrf2 did not reduce UBC transcription under either basal or stressful conditions, but significantly impaired the constitutive and inducible expression of well‐known antioxidant response element‐dependent genes. A chromatin immunoprecipitation assay consistently failed to detect Nrf2 binding to the UBC promoter sequence. By contrast, depletion of HSF1, but not HSF2, significantly compromised stress‐induced UBC expression. Critically, HSF1‐mediated UBC trans‐activation upon arsenite exposure relies on transcription factor binding to previously mapped distal HSEs, as demonstrated to occur under proteasome inhibition. These data highlight HSF1 as the pivotal transcription factor that translates different stress signals into UBC gene transcriptional induction. [ABSTRACT FROM AUTHOR]

Ataxia telangiectasia (A-T) is a rare incurable neurodegenerative disease caused by biallelic mutations in the gene for ataxia-telangiectasia mutated ( ATM). The lack of a functional ATM kinase leads to a pleiotropic phenotype, and oxidative stress is considered to have a crucial role in the complex physiopathology. Recently, steroids have been shown to reduce the neurological symptoms of the disease, although the molecular mechanism of this effect is largely unknown. In the present study, we have demonstrated that dexamethasone treatment of A-T lymphoblastoid cells increases the content of two of the most abundant antioxidants [glutathione ( GSH) and NADPH] by up to 30%. Dexamethasone promoted the nuclear accumulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 to drive expression of antioxidant pathways involved in GSH synthesis and NADPH production. The latter effect was via glucose 6-phosphate dehydrogenase activation, as confirmed by increased enzyme activity and enhancement of the pentose phosphate pathway rate. This evidence indicates that glucocorticoids are able to potentiate antioxidant defenses to counteract oxidative stress in ataxia telangiectasia, and also reveals an unexpected role for dexamethasone in redox homeostasis and cellular antioxidant activity. [ABSTRACT FROM AUTHOR]

The possibility to clone, express and purify recombinant enzymes have originated the opportunity to dispose of a virtually infinite array of proteins that could be used in the clinics to treat several inherited and acquired pathological conditions. However, the direct administration of these recombinant proteins faces some intrinsic difficulties, such as degradation by circulating proteases and/or inactivation by the patient immune system. The use of drug delivery systems may overcome these limitations. Concerning recombinant enzyme therapy, the present review will mainly focus on the exploitation of erythrocytes as a carrier system for enzymes removing potentially noxious metabolites from the circulation, either as limiting treatment strategy for auxotrophic tumours or as a detoxing approach for some intoxication type inherited metabolic disorders. Moreover, the possibility of using RBCs as a potential delivering system addressing the enzymes to the monocyte-macrophages of reticular endothelial system for the treatment of diseases associated with this cell lineage, e.g. lysosome storage diseases, will be briefly discussed. [ABSTRACT FROM AUTHOR]

The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. [ABSTRACT FROM AUTHOR]

Photobacteriosis or fish pasteurellosis is a bacterial disease affecting wild and farm fish. Its etiological agent, the gram negative bacterium Photobacterium damselae subsp. piscicida, is responsible for important economic losses in cultured fish worldwide, in particular in Mediterranean countries and Japan. Efforts have been focused on gaining a better understanding of the biology of the pathogenic microorganism and its natural hosts with the aim of developing effective vaccination strategies and diagnostic tools to control the disease. Conventional vaccinology has thus far yielded unsatisfactory results, and recombinant technology has been applied to identify new antigen candidates for the development of subunit vaccines. Furthermore, molecular methods represent an improvement over classical microbiological techniques for the identification of P. damselae subsp. piscicida and the diagnosis of the disease. The complete sequencing, annotation, and analysis of the pathogen genome will provide insights into the pathogen laying the groundwork for the development of vaccines and diagnostic methods. [ABSTRACT FROM AUTHOR]

Mycobacterium avium is an intracellular pathogen preferentially infecting human macrophages where they activate the JAK/STAT1 pathway. This activation enhances the survival of infected cells, but, at the same time, makes macrophages optimal targets for drugs development against p-tyr701stat1. In this study, we demonstrate that the fast and transient activity of the JAK/STAT1 pathway occurs immediately after macrophages internalization of heat-killed M. avium or inert particles. Furthermore, we show that a persistent Stat1 pathway activation occurs only when an intracellular M. avium infection is established in macrophages. These results strongly indicate different mechanisms of p-tyr701Stat1 activation. In particular, here we report findings aiming at explaining the short-time enhancement of p-tyr701Stat1 and shows its predominant relationship with FcγRs engagement during the internalization process. Furthermore, we demonstrate that opsonized live M. avium is phagocytosed by macrophages involving membrane receptors not related with JAK/STAT1 signalling pathway. On the contrary, heat-inactivated bacilli or latex particles seem to be internalized only after involvement of FcγRs and subsequent Stat1 phosphorylation. [ABSTRACT FROM AUTHOR]

Drug delivery is a growing field of interdisciplinary activities that combine the use of new materials with the biochemical properties of selected drugs, with the aim of improving their therapeutic action and reducing their toxicity. In few cases, proper medical devices have been also realized to implement new drug delivery modalities. In this article, we have summarized available information and our experience on the use of autologous Red Blood Cells as carriers for drugs to be released within the vascular system. This is not a comprehensive review, but it focusses on the mechanisms that are available to distribute drugs in circulation by carrier red blood cells and provide illustrative examples on how this is currently obtained. We have not included a summary of clinical data collected in recent years using this technology but simply provided proper references for the interested readers. Finally, a special attention is devoted to the possibility of entrapping, into autologous red blood cells, recombinant drug-binding proteins. This new strategy is opening the way at a new modality to influence the vascular distribution of drugs by realizing a dynamic circulating container (the engineered red cell) capable of reversible binding and transportation of one or more drugs of interest selected on the bases of the red cell entrapped target proteins. This new modality is not yet fully developed and explored but will certainly provide a technical solution to the problem of stabilizing drug concentration in circulation improving drug efficacy and reducing drug toxicity. © 2011 IUBMB IUBMB Life, 2011 [ABSTRACT FROM AUTHOR]

. High-biomass blooms of A. taylori Balech have recently been spreading over new Mediterranean areas, with evident adverse effects on the marine ecosystem. In 1999-2000, a new Mediterranean locality was affected by blooms of A. taylori: the West Bay of Vulcano (Aeolian Islands, Tyrrhenian Sea), with maximum cell densities of 1.2 × 107 cells 1−1 in August 1999 and 4.0 × 106 cells 1−1 in August 2000 observed together with yellowish water discoloration. The seawater samples contained high concentrations of nutrients as DIN (Dissolved Inorganic Nitrogen), especially NH3-N with values of 14.4 μM and TOT-P (Total Phosphorus) with values of 3.2 μM due to the anthropic presence and discharge of untreated sewage. The climatic conditions also seem to influence the occurrence and spreading of the A. taylori blooms in the Vulcano Bay. Clonal cultures of A. taylori, established from Italian and Spanish seawater samples, were used for the sequence analyses of the 5.8S rDNA gene and ITS regions in order to study the genetic variability of different geographical populations of Alexandrium species in the Mediterranean area and to further develop the molecular markers for HAB key-species. [ABSTRACT FROM AUTHOR]

PCR primers targeting the internal transcribed spacer (ITS)-5.8S rDNA regions specific for the genus Alexandrium were used to develop an ELISA assay method to detect and enumerate this genus in cultured isolates. The solid-phase ELISA involves the application of a biotinylated labeled primer to target the specific ITS-5.8S rDNA region; the PCR-amplified products, generated in the presence of digoxigenin-11-deoxiuracil triphosphate nucleotide, are captured on the streptavidin-coated microplate. The captured molecules were hybridized to an anti-digoxigenin antibody conjugated with alkaline phosphatase. The presence and number of the Alexandrium cells in the samples resulted in a proportional appearance of color generated by the phosphatase activity in the presence of a chromogenic substrate and measured in a plate reader. This PCR and immunoassay solid-phase assay proved to be a useful technique to detect the presence of Alexandrium sp. in cultured isolates and seawater samples. [ABSTRACT FROM AUTHOR]

Examines the ubiquitinated repeats in human erythroid α-spectrin. Importance of deformability and resistance to mechanical stress by the red cells; Susceptibility of the ubiquitination; Effects of mutation on the interaction resulting in the increase of membrane fragility.

Focuses on the identification of dinophyceae, Alexandrium, species using polymerase chain reaction (PCR) and rDNA-targeted probes. Amplification of the internal transcribed spacers and the ribosomal RNA gene; Cross-reactivity of the PCR primers and probe.

Demonstrates the effects of the activation of the ubiquitin proteolytic system on IkappaBalpha in the lymph nodes of animals infected with murine acquired immunodeficiency syndrome. Proteosomal activity; Reduction in the level of IkappaBalpga in lymph nodes of infected mice; Absence of effect of IkappaBalpha degradation on nuclear factor-kappaB DNA binding activity.

Examines the alteration of α-spectrin ubiquitination due to age-dependent changes in erythrocyte membrane. Decrease of cell age-dependence; Dependence of ubiquitination on the source of the red cell membranes; Significance of membrane architecture for the decrease of age-dependent in ubiquitination.

K48R ubiquitin (K48R-Ub) is an analogue of native ubiquitin that does not form polyubiquitin chain conjugates. Targeted delivery of this recombinant mutant ubiquitin to human macrophages results in an intracellular increase in the ubiquitin analogue. IkBα polyubiquitination and degradation were significantly inhibited in K48R-Ub targeted macrophages upon stimulation with lipopolysaccharide. The ability to reduce IkBα degradation was also associated with a reduced production of TNF-α, the gene of which is under NF-kB control. At a concentration of 0.1 μM, dexamethasone was less effective than K48R-Ub in preventing IkBα depletion and TNF-α release. These data suggest that ubiquitin analogues are potent suppressors of TNF-α release in macrophages. [ABSTRACT FROM AUTHOR]

Demonstrates that in murine AIDS, the adenosine triphosphate-dependent and ubiquitin-dependent proteolytic system is strongly affected at least in the lymph nodes of infected mice. Features of retrovirus-induced murine AIDS including lymphoproliferation, chronic B-cell activation resulting in hypergammaglobulinemia and profound immunodeficiency; Coincidence of the accumulation of ubiquitin conjugates and induction of the conjugating system with an increase in the proteolytic activity.

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with 125I-labeled ubiquitin. A few proteins able to form thiol esters with 125I-Ub and probably corresponding to ubiquitin-conjugating enzymes, El and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state. [ABSTRACT FROM AUTHOR]

Recently optimized technologies that permit the reversible opening of nanopores across the red blood cell membrane, give the extraordinary opportunity for reengineering human erythrocytes to be used in different biomedical applications, both for therapeutic and diagnostic purposes. Engineered erythrocytes have been exploited as a system for the controlled release of drugs in circulation upon encapsulation of prodrugs or small molecules; as bioreactors when they are endowed of recombinant enzymes able to catalyze the conversion of toxic metabolite into inert products; as drug targeting system for the delivery of compounds to the reticuloendothelial system inducing proper senescent signals on the drug-loaded erythrocyte membrane; as carrier of contrasting agents for diagnostic procedures. Preclinical development of these different applications has taken advantage from the use of proper animal models whose erythrocytes can be reengineered as the human ones or the encapsulation procedures can be adapted on the basis of their specific erythrocyte biological features. Successful results, obtained both in vitro and in preclinical studies, have prompted several clinicians to start pilot clinical investigations in different conditions and some new companies to start the industrialization of selected loading technologies and to initiate clinical development programs. This short review summarizes the key features that, to the best of our knowledge, have been crucial to advance the products toward regulatory clinical approval making reengineering of erythrocytes a modality to treat patients with limited or absent therapeutic options. WIREs Nanomed Nanobiotechnol 2017, 9:e1454. doi: 10.1002/wnan.1454 For further resources related to this article, please visit the . [ABSTRACT FROM AUTHOR]

Galactokinase (GALK1) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet. Galactokinase deficiency results from mutation in the GALK1 gene mapped on 17q24. Since GK1 cDNA was cloned about 20 mutations (prevalently deletions and missense) have been reported to date. Most of these reported mutations are confined to single families, and only one of them, P28T, has been referred as the founder Romani mutation. In this paper we report two novel missense mutations in GALK1 gene, identified in two unrelated patients with galactokinase deficiency. One mutation, g.575G>A, substitutes a valine for a methionine at amino acid 32 (p.V32M), while the other mutation, g.2839G>A, results in the arginine to glutamine substitution p.R239Q (GenBank sequence L76927). Biochemical studies demonstrate that these mutations led to a drastic modification in GALK activity when individual mutant cDNAs were expressed in an E. coli system. These findings indicate the pathogeneticity of these mutations causing GALK deficiency. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Discusses the monitoring of erythropoietin (rEpo) abuse in athletes. Reason for using erythropoietin; Details on threshold values that indicate rEpo abuse; Increase in hematocrits during treatment.

The aim of our work was to develop a new molecular method for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 directly in milk samples. Three specific target sequences were chosen for a multiplex polymerase chain reaction (PCR) assay: a 155-bp region of the Salmonella spp. tetrathionate reductase (ttr ) locus; a 173-bp region of the L. monocytogenes listeriolysin O gene (hlyA ); a 217-bp region of the E. coli O157 lipopolysaccharide gene (rfbE ). An internal amplification control was also included to detect PCR inhibition. In addition, a magnetic-based extraction method was also developed to isolate PCR-ready DNA from milk. The assay was able to detect, whether alone or mixed, also with a difference of 2 log units, as few as 102 cells of each pathogen per 10 mL of spiked milk. PRACTICAL APPLICATIONS Escherichia coli O157, Salmonella spp. and Listeria monocytogenes are among the most dangerous foodborne bacterial pathogens in terms of human health and disease, and their detection through current microbiological culture methods involves considerable spending of time and effort. Therefore, the development of rapid and sensitive diagnostic methods is needed. The magnetic DNA extraction proposed in this study concentrates the target organism DNA, removes the inhibitors and presents the additional advantages of being safe and suitable for automation systems. Moreover, the developed multiplex polymerase chain reaction (mPCR) enables the simultaneous detection of more than one target sequence in the same reaction, producing considerable cost and reagent savings. The assay, combining magnetic extraction and mPCR, has proven to be a valuable and cost-effective tool for monitoring the presence of these pathogens directly in milk samples. Therefore, it could provide a major contribution to efficiency and logistics of molecular diagnostics. [ABSTRACT FROM AUTHOR]