Grzegorz Węgrzyn

Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are inherited, severe, progressive, metabolic disorders caused by deficiencies in different enzymes involved in degradation of glycosaminoglycans (GAGs). Although enzyme replacement therapy (ERT) has recently been available for MPS type I, and clinical trials have been performed in ERT for MPS II and MPS VI, there is little chance that this kind of treatment may be effective for neurodegenerative forms of MPS (due to inefficient delivery of enzymes to central nervous system through the blood–brain barrier), hence currently there is no effective therapy available for them. Therefore, we aim to develop an alternative therapy for these diseases. We found that genistein (4′,5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) inhibits synthesis of GAGs considerably in cultures of fibroblasts of MPS patients (types I, II, IIIA and IIIB were tested). Prolonged cultivation of these cells in the presence of genistein resulted in reduction of GAG accumulation and normalization of cells as estimated by biochemical tests and electron microscopic analysis, respectively. As genistein inhibits kinase activity of epidermal growth factor receptor, which is required for full expression of genes coding for enzymes involved in GAG production, we propose to consider a substrate reduction therapy for MPS, which is referred to as ‘gene expression-targeted isoflavone therapy’.

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Functional domains of DnaA proteins

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Genistein Improves Neuropathology and Corrects Behaviour in a Mouse Model of Neurodegenerative Metabolic Disease

Background Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

Bacterial replication initiator DnaA. Rules for DnaA binding and rolesof DnaA in origin unwinding and helicase loading

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

BackgroundAlthough understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids.ResultsEscherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin) or plasmid pOri2 (with double ColE1 replication origins), were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate (PP) pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG), the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA.ConclusionThe presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

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.

Genetic Switches During Bacteriophage λ Development

Publisher Summary This chapter discusses recent discoveries in this field, with special emphasis on molecular systems operating to sense the current status of the host physiology and to switch bacteriophage development into potentially the most effective pathway. The development of bacteriophage λ must be precisely controlled in response to environmental conditions and the physiological state of the host cell. Such a control, performed at the genetic level, ensures choosing an optimal developmental strategy for successful propagation of the virus. This chapter have distinguished five genetic switches in bacteriophage λ development (Ag43 phase variation, the ‘‘lysis versus lysogenization’’ decision, prophage induction, a change from early to late DNA replication mode, and induction of the host cell lysis) whose molecular mechanisms can be proposed. These processes include repression and activation of transcription initiation, antitermination of transcription, general and site-specific recombination, a role for chaperone proteins in macromolecular assembly and DNA replication, and even the control of development.

Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

ABSTRACT For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyimutants resistant to neomycin. We constructed genetically modifiedV. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.