Magdalena Narajczyk

Genistein in Sanfilippo disease: a randomized controlled crossover trial.

Sanfilippo disease (mucopolysaccharidosis type III [MPS III]) is a rare neurodegenerative metabolic disease caused by a deficiency of 1 of the 4 enzymes involved in the degradation of heparan sulfate (HS), a glycosaminoglycan (GAG). Genistein has been proposed as potential therapy but its efficacy remains uncertain. We aimed to determine the efficacy of genistein in MPS III.

Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses

Sanfilippo disease (mucopolysaccharidosis type III, MPS III) is a severe metabolic disorder caused by accumulation of heparan sulfate (HS), one of glycosaminoglycans (GAGs), due to a genetic defect resulting in a deficiency of GAG hydrolysis. This disorder is characterized as the most severe neurological form of MPS, revealing rapid deterioration of brain functions. Among therapeutic approaches for MPS III, one of the most promising appears to be the substrate reduction therapy (SRT). Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is an isoflavone that has been used in SRT for MPS III. In this report, we tested effects of other flavonoids (apigenin, daidzein, kaempferol and naringenin) on GAG synthesis. Their cytotoxicity and anti-proliferation features were also tested. We found that daidzein and kaempferol inhibited GAG synthesis significantly. Moreover, these compounds were able to reduce lysosomal storage in MPS IIIA fibroblasts. Interestingly, although genistein is believed to inhibit GAG synthesis by blocking the tyrosine kinase activity of the epidermal growth factor receptor, we found that effects of other flavonoids were not due to this mechanism. In fact, combinations of various flavonoids resulted in significantly more effective inhibition of GAG synthesis than the use of any of these compounds alone. These results, together with results published recently by others, suggest that combination of flavonoids can be considered as a method for improvement of efficiency of SRT for MPS III.

Why are behaviors of children suffering from various neuronopathic types of mucopolysaccharidoses different

Mucopolysaccharidoses (MPS) are inherited metabolic disorders from the group of lysosomal storage diseases (LSD). They arise from mutations causing dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in lysosomes. Impaired degradation of these compounds results in their accumulation in cells and dysfunction of most tissues and organs of patients. If heparan sulfate (HS) is the sole or one of stored GAGs, brain functions are also affected. However, despite the fact that products of incomplete degradation of the same chemical, HS, are accumulated in brains of patients suffering from Hurler disease (MPS type I), Hunter disease (MPS type II), Sanfilippo disease (MPS type III) and Sly disease (MPS type VII), and obvious deterioration of brain functions occur in these patients, their behavior is considerably different between various types of MPS. Here we asked the question about biochemical reasons of these differences. We performed theoretical analysis of products of incomplete HS degradation that accumulate in tissues of patients diagnosed for these diseases. A correlation between chemical structures of incompletely degraded HS and behaviors of patients suffering from particular MPS types was found. We propose a hypothesis that particular chemical moieties occurring at the ends of incompletely degraded HS molecules may determine characteristic behavioral disturbances, perhaps due to chemical reactions interfering with functions of neurons in the brain. A possible experimental testing of this hypothesis is also proposed. If the hypothesis is true, it might shed some new light on biochemical mechanisms of behavioral problems occurring not only in MPS but also in some other diseases.

The Use of Elevated Doses of Genistein-Rich Soy Extract in the Gene Expression-Targeted Isoflavone Therapy for Sanfilippo Disease Patients

Mucopolysaccharidoses (MPS) are severe, inherited metabolic disorders caused by storage of glycosaminoglycans (GAGs). Sanfilippo disease (mucopolysaccharidosis type III, MPS III) is described as severe neurological type of MPS, characterized by rapid deterioration of brain functions. No therapy for Sanfilippo disease is approved to date, however, a specific substrate reduction therapy (SRT), called gene expression-targeted isoflavone therapy (GET IT), has been used as an experimental therapy. In this report, we describe effects of treatment of six Sanfilippo disease patients with GET IT, in which the dose of genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), an active compound of GET IT present in the soy isoflavone extract, has been increased to 10, and then to 15 mg/kg/day, contrary to the previously reported dose of 5 mg/kg/day. By measuring levels of urinary GAGs and assessing hair dysmorphology as biomarkers, and by considering clinical symptoms of patients, we obtained results suggesting that elevated doses of genistein may improve efficacy of GET IT for Sanfilippo disease.

Substrate reduction therapies for mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic disorders, caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in lysosomes. Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs, including the heart, respiratory system, bones, joints and central nervous system. Depending on the kind of deficient enzyme, 11 types and subtypes of MPS are currently recognized. Although enzyme replacement therapy has been developed for 3 types of MPS (types I, II and VI), this treatment was found to be effective only in management of somatic symptoms. Since all MPS types except IVA, IVB and VI are characterized by various problems with functioning of the central nervous system (CNS), a search for effective treatment of this system is highly desirable. Recent discoveries suggested that substrate reduction therapy may be an efficient method for treatment of MPS patients, including their CNS. In this review, different variants of this therapy will be discussed in the light of recently published reports.

Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage

A large scale analysis presented in this article focuses on biological and physiological variety of bacteriophages. A collection of 83 bacteriophages, isolated from urban sewage and able to propagate in cells of different bacterial hosts, has been obtained (60 infecting Escherichia coli, 10 infecting Pseudomonas aeruginosa, 4 infecting Salmonella enterica, 3 infecting Staphylococcus sciuri, and 6 infecting Enterococcus faecalis). High biological diversity of the collection is indicated by its characteristics, both morphological (electron microscopic analyses) and biological (host range, plaque size and morphology, growth at various temperatures, thermal inactivation, sensitivity to low and high pH, sensitivity to osmotic stress, survivability upon treatment with organic solvents and detergents), and further supported by hierarchical cluster analysis. By the end of the research no larger collection of phages from a single environmental source investigated by these means had been found. The finding was confirmed by whole genome analysis of 7 selected bacteriophages. Moreover, particular bacteriophages revealed unusual biological features, like the ability to form plaques at low temperature (4 °C), resist high temperature (62 °C or 95 °C) or survive in the presence of an organic solvents (ethanol, acetone, DMSO, chloroform) or detergent (SDS, CTAB, sarkosyl) making them potentially interesting in the context of biotechnological applications.

Genomic, Proteomic and Morphological Characterization of Two Novel Broad Host Lytic Bacteriophages ΦPD10.3 and ΦPD23.1 Infecting Pectinolytic Pectobacterium spp. and Dickeya spp.

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.

Switch from θ to σ replication of bacteriophage λ DNA: factors involved in the process and a model for its regulation

Bacteriophage λ genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling λ DNA replication may be of bio-medical importance. During lytic development of bacteriophage λ, its genome is replicated according to the θ (circle-to-circle) mode early after infection, and then it is switched to the σ (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of λ DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the λO initiator protein, another suggested a crucial role for transcriptional activation of the oriλ region and factors involved in the control of the pR promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of λ DNA replication and appearance of σ replication intermediates. On the other hand, regulators affecting activity of the pR promoter (responsible for initiation of transcription, which activates oriλ) directly or indirectly influenced directionality of λ DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of oriλ, rather than transient impairment of the λO proteolysis, is responsible for the control of the switch from θ to σ replication, and propose a model for this control.

Synthetic genistein derivatives as modulators of glycosaminoglycan storage

BackgroundMucopolysaccharidoses (MPS) are severe metabolic disorders caused by accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential treatment procedures of MPS. Importantly, small molecules used in such a therapy might potentially cross the blood–brain barrier (BBB) and improve neurological status of patients, as reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4 H-1-benzopyran-4-one, also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to develop a set of synthetically modified genistein molecules and characterize physicochemical as well as biological properties of these compounds.MethodsFollowing parameters were determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB.ResultsWe observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation. Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than the natural isoflavone.ConclusionSome compounds tested in this study might be promising candidates for further studies on therapeutic agents in MPS types with neurological symptoms.

Synthesis of antimicrobial silver nanoparticles through a photomediated reaction in an aqueous environment.

A fast, economical, and reproducible method for nanoparticle synthesis has been developed in our laboratory. The reaction is performed in an aqueous environment and utilizes light emitted by commercially available 1 W light-emitting diodes (λ =420 nm) as the catalyst. This method does not require nanoparticle seeds or toxic chemicals. The irradiation process is carried out for a period of up to 10 minutes, significantly reducing the time required for synthesis as well as environmental impact. By modulating various reaction parameters silver nanoparticles were obtained, which were predominantly either spherical or cubic. The produced nanoparticles demonstrated strong antimicrobial activity toward the examined bacterial strains. Additionally, testing the effect of silver nanoparticles on the human keratinocyte cell line and human peripheral blood mononuclear cells revealed that their cytotoxicity may be limited by modulating the employed concentrations of nanoparticles.