The Role of Dimethyl Sulfoxide (DMSO) in Gene Expression Modulation and Glycosaminoglycan Metabolism in Lysosomal Storage Disorders on an Example of Mucopolysaccharidosis

Obstacles to effective therapies for mucopolysaccharidoses (MPSs) determine the need for continuous studies in order to enhance therapeutic strategies. Dimethyl sulfoxide (DMSO) is frequently utilised as a solvent in biological studies, and as a vehicle for drug therapy and the in vivo administration of water-insoluble substances. In the light of the uncertainty on the mechanisms of DMSO impact on metabolism of glycosaminoglycans (GAGs) pathologically accumulated in MPSs, in this work, we made an attempt to investigate and resolve the question of the nature of GAG level modulation by DMSO, the isoflavone genistein solvent employed previously by our group in MPS treatment. In this work, we first found the cytotoxic effect of DMSO on human fibroblasts at concentrations above 3%. Also, our results displayed the potential role of DMSO in the regulation of biological processes at the transcriptional level, then demonstrated a moderate impact of the solvent on GAG synthesis. Interestingly, alterations of lysosomal ultrastructure upon DMSO treatment were visible. As there is growing evidence in the literature that DMSO can affect cellular pathways leading to numerous changes, it is important to expand our knowledge concerning this issue

Metal and antibiotic resistance of bacteria isolated from the Baltic Sea

The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzedand the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78 %), while Alphaproteobacteria (8 %), Actinobacteria (10 %), and Bacteroidetes (4 %) were lessabundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the abilityto produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relationbetween resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not tospectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as anaminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance tosome antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycinand tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effectsof NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrationsof Mn2+ due to chemical interactions that potentially lead to precipitation. [Int Microbiol 2012; 15(3):131-139

Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier\ud (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

Female Fabry disease patients and X-chromosome inactivation

Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α- galactosidase A (GLA). Once it was thought to affect only hemizygous males. Over the last fifteen years, research has shown that most females carrying mutated allele also develop symptoms, demonstrating a wide range of disease severity, from a virtually asymptomatic to more classical profile, with cardiac, renal, and cerebrovascular manifestations. This variable expression in females is thought to be influenced by the process of X-chromosome inactivation (XCI). The aim of this study was to assess severity of the clinical phenotype, to analyze XCI patterns, and to estimate their effect on disease manifestation in twelve female Fabry disease patients from five unrelated Polish families. Our analyses revealed that patients presented with the broad range of disease expression - from mild to severe, and their clinical involvement did not correlate with XCI profiles. Female carriers of the mutation in the GLA gene with the random XCI may present with the wide range of disease signs and symptoms. Thus, XCI is not a main factor in the phenotype variability of Fabry disease manifestation in heterozygous females

Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also inMPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease inMTOR transcript level was observed at these conditions

Lysosome Alterations in the Human Epithelial Cell Line HaCaT and Skin Specimens: Relevance to Psoriasis

Despite the constantly updated knowledge regarding the alterations occurring in the cells of patients with psoriasis, the status and the role of the lysosome, a control center of cell metabolism, remain to be elucidated. The architecture of the epidermis is largely regulated by the action of lysosomes, possibly activating signaling pathways in the cellular crosstalk of keratinocytes—epidermal cells—with infiltrating immune cells. Thus, in the present study, lysosome alterations were examined in vitro and in situ using a two-dimensional (2D) keratinocyte model of HaCaT cells with “psoriasis-like” inflammation and skin specimens, respectively. Specific fluorescence and immunohistochemical staining showed an augmented level of acidic organelles in response to keratinocyte activation (mimicking a psoriatic condition while maintaining the membrane integrity of these structures) as compared with the control, similar to that seen in skin samples taken from patients. Interestingly, patients with the most pronounced PASI (Psoriasis Area and Severity Index), BSA (Body Surface Area), and DLQI (Dermatology Life Quality Index) scores su�ered a high incidence of positive lysosomal-associated membrane protein 1 (LAMP1) expression. Moreover, it was found that the gene deregulation pattern was comparable in lesioned (PP) and non-lesioned (PN) patient-derived skin tissue, which may indicate that these alterations occur prior to the onset of the characteristic phenotype of the disease. Changes in the activity of genes encoding the microphthalmia family (MiT family) of transcription factors and mammalian target of rapamycin complex 1 (MTORC1) were also observed in the in vitro psoriasis model, indicating that the biogenesis pathway of this arm is inhibited. Interestingly, in contrast to the keratinocytes of HaCaT with “psoriasis-like” inflammation, LAMP1 was up-regulated in both PP and PN skin, which can be a potential sign of an alternative mechanism of lysosome formation. Defining the molecular profile of psoriasis in the context of “the awesome lysosome” is not only interesting, but also desired; therefore, it is believed that this paper will serve to encourage other researchers to conduct further studies on this subject

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

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