Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway

<p>Abstract</p> <p>Background</p> <p>Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome).</p> <p>Methods</p> <p>To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates.</p> <p>Results</p> <p>Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17β-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts.</p> <p>Conclusion</p> <p>The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.</p

Synthetic genistein derivatives as modulators of glycosaminoglycan synthesis

Background: Mucopolysaccharidoses (MPS) are severe metabolic disorders caused by 26 accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain 27 lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential 28 treatment procedures of MPS. Importantly, small molecules used in such a therapy might 29 potentially cross the blood-brain barrier (BBB) and improve neurological status of patients, as 30 reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one, 31 also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to 32 the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to 33 develop a set of synthetically modified genistein molecules and characterize physicochemical as 34 well as biological properties of these compounds. Methods: Following parameters were 35 determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell 36 proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s 37 tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB. Results: We 38 observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more 39 efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The 40 tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation. 41 Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than 42 the natural isoflavone. Conclusion: Some compounds tested in this study might be promising 43 candidates for further studies on therapeutic agents in MPS types with neurological symptoms

Heat Treatment Effect on Eu 3+

Glass systems of 73TeO2-4BaO-3Bi2O3-2Eu2O3-xAg (in molar ratio where x = 0, 1, 2, and 3) compositions have been successfully synthesized. Silver nanoparticles were obtained with the employment of heat treatment (HT) procedure executed at 350°C. Glass transition temperatures of different compositions have been determined through DSC measurements. XRD results presented characteristic amorphous halo indicating lack of long range order in the samples. FTIR structural studies revealed that glass matrix is mainly composed of TeO3 and TeO4 species and is stable after different applied heat treatment times. X-ray photoelectron spectroscopy (XPS) measurements confirmed that in selected samples part of Ag ions changed oxidation state to form Ag0 species. TEM measurements revealed nanoparticles of size in the range of 20–40 nm. UV-vis absorption results demonstrated characteristic transitions of Eu3+ ions. Additionally, UV-vis spectra of samples heat-treated for 6, 12, 24, and 48 hours presented bands related to silver nanoparticles. Photoluminescence (PL) studies have been performed with excitation wavelength of λexc=395 nm. Obtained spectra exhibited peaks due to 5D0-7FJ (where J=2,3,4) and 5D1-7FJ (where J=1,2,3) transitions of Eu3+. Moreover, luminescence measurement indicated enhancement of rare earth ions emissions in several of the annealed samples. Increase of emission intensity of about 35% has been observed

CRP-binding bacteriophage as a new element of layer-by-layer assembly carbon nanofiber modified electrodes

Recently, bacteriophage particles have started to be applied as a new biomaterial for developing sensing platforms. They can be used as both a recognition element or/and as building blocks, template/scaffold. In this paper, we studied a bacteriophage selected through phage-display technology. The chosen bacteriophage acted as a building block for creating a carbon nanofiber-based electrode and as a new receptor/binding element that recognizes C-reactive protein (CRP) – one of the markers of inflammatory processes in the human body. The binding efficiency of the selected phage towards CRP is two orders of magnitude higher than in the wild type. We demonstrate that the phage-based sensor is selective against other proteins. Finally, we show that layer-by-layer methods are suitable for deposition of negatively charged phages (wild or CRP-binding) with positively charged carbon nanofibers for electrode surface modification. A three-layered electrode was successfully used for molecular recognition of CRP, and the molecular interactions were studied using electrochemical, biological, and optical methods, including microscopic and spectroscopic analyses

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

Experimental Plant Biology: Why Not?! Genistein: a natural isoflavone with a potential for treatment of genetic diseases

Abstract Genistein [4 ,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is a natural isoflavone occurring in many plants known to possess various biological activities, ranging from phyto-oestrogenic to antioxidative actions. Recent studies indicated that this isoflavone can also be considered as a drug for as yet untreatable genetic diseases. In the present review, we discuss a plausible use of genistein in treatment of two genetic disorders: CF (cystic fibrosis) and MPS (mucopolysaccharidosis). Although various biological actions of genistein are employed in these two cases, in vitro studies, tests on animal models and pilot clinical trials suggest that this plant-derived compound might be a real hope for patients suffering from severe inherited disorders with relatively complicated pathomechanisms, including those affecting the central nervous system

Experimental Plant Biology: Why Not?! Genistein: a natural isoflavone with a potential for treatment of genetic diseases

Abstract Genistein [4 ,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is a natural isoflavone occurring in many plants known to possess various biological activities, ranging from phyto-oestrogenic to antioxidative actions. Recent studies indicated that this isoflavone can also be considered as a drug for as yet untreatable genetic diseases. In the present review, we discuss a plausible use of genistein in treatment of two genetic disorders: CF (cystic fibrosis) and MPS (mucopolysaccharidosis). Although various biological actions of genistein are employed in these two cases, in vitro studies, tests on animal models and pilot clinical trials suggest that this plant-derived compound might be a real hope for patients suffering from severe inherited disorders with relatively complicated pathomechanisms, including those affecting the central nervous system

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

Quantitative estimation of lysosomal storage in mucopolysaccharidoses by electron microscopy analysis

Mucopolysaccharidoses (MPS) are severe inherited metabolic disorders caused by storage of glycosaminoglycans (GAGs). The level of accumulated GAGs is an important parameter in assessment of the severity of the disease and the efficacy of treatment; unfortunately, biochemical methods are often unreliable and only semi-quantitative. Therefore, finding other methods for estimation of GAG levels and/or assessment of the efficacy of applied therapy is very important. Although monitoring of GAG levels during therapy is crucial, in this work it is proposed that analysis of the ultrastructure of MPS cells by electron microscopic techniques can be considered as an alternative and reliable method for assessment of lysosomal storage. The number of complex lysosomal structures was found to be significantly higher in MPS cells relative to controls, while it decreased significantly in response to either enzyme replacement therapy or substrate reduction therapy. Thus, this parameter, easily assessed by electron microscopy, appears to correspond to the physiological state of MPS cells

CRP-binding bacteriophage as a new element of layer-by-layer assembly carbon nanofiber modified electrodes

Recently, bacteriophage particles have started to be applied as a new biomaterial for developing sensing platforms. They can be used as both a recognition element or/and as building blocks, template/scaffold. In this paper, we studied a bacteriophage selected through phage-display technology. The chosen bacteriophage acted as a building block for creating a carbon nanofiber-based electrode and as a new receptor/binding element that recognizes C-reactive protein (CRP) – one of the markers of inflammatory processes in the human body. The binding efficiency of the selected phage towards CRP is two orders of magnitude higher than in the wild type. We demonstrate that the phage-based sensor is selective against other proteins. Finally, we show that layer-by-layer methods are suitable for deposition of negatively charged phages (wild or CRP-binding) with positively charged carbon nanofibers for electrode surface modification. A three-layered electrode was successfully used for molecular recognition of CRP, and the molecular interactions were studied using electrochemical, biological, and optical methods, including microscopic and spectroscopic analyses