Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box-Behnken design to model and optimize the ...parameters of β-galactosidase entrapment in sol-gel-derived silica composites. We have also demonstrated the influence of polymer-polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on β-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum β-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of β-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.
Specific nucleoprotein complexes are formed strictly to prevent over-initiation of DNA replication. An example of those is the so-called handcuff complex, in which two plasmid molecules are coupled ...together with plasmid-encoded replication initiation protein (Rep). In this work, we elucidate the mechanism of the handcuff complex disruption. In vitro tests, including dissociation progress analysis, demonstrate that the dimeric variants of plasmid RK2 replication initiation protein TrfA are involved in assembling the plasmid handcuff complex which, as we found, reveals high stability. Particular proteases, namely Lon and ClpAP, disrupt the handcuff by degrading TrfA, thus affecting plasmid stability. Moreover, our data demonstrate that TrfA monomers are able to dissociate handcuffed plasmid molecules. Those monomers displace TrfA molecules, which are involved in handcuff formation, and through interaction with the uncoupled plasmid replication origins they re-initiate DNA synthesis. We discuss the relevance of both Rep monomers and host proteases for plasmid maintenance under vegetative and stress conditions.
Nucleoid-associated proteins (NAPs) are responsible for maintaining highly organized and yet dynamic chromosome structure in bacteria. The genus Mycobacterium possesses a unique set of NAPs, ...including Lsr2, which is a DNA-bridging protein. Importantly, Lsr2 is essential for the M. tuberculosis during infection exhibiting pleiotropic activities including regulation of gene expression (mainly as a repressor). Here, we report that deletion of lsr2 gene profoundly impacts the cell morphology of M. smegmatis, which is a model organism for studying the cell biology of M. tuberculosis and other mycobacterial pathogens. Cells lacking Lsr2 are shorter, wider, and more rigid than the wild-type cells. Using time-lapse fluorescent microscopy, we showed that fluorescently tagged Lsr2 forms large and dynamic nucleoprotein complexes, and that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Moreover, lsr2 deletion exerts a significant effect on the replication time and replisome dynamics. Thus, we propose that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA and therefore influencing mycobacterial cell cycle.
Breast cancer is one of cancer’s most deadly varieties. Its variability makes the development of personalized therapies very difficult. Therefore, improvement of classic chemotherapy is still one of ...the important challenges of cancer research. We addressed this issue applying nanotechnology to verify the influence of silver nanoparticles (AgNPs) on doxorubicin (DOX) anticancer activity and assess if the size of AgNPs affects their interactions with DOX. We employed a broad spectrum of biophysical methods, characterizing 5 and 50 nm AgNPs interactions with DOX using UV–Vis spectroscopy, dynamic light scattering, fluorescence spectroscopy, and atomic force microscopy imaging. Biological effects of observed AgNPs-DOX interactions were assessed utilizing MTT and 3D Matrigel assays on SKBR3 and MDA-MB-231 breast cancer cell lines. Obtained results indicate direct interactions between AgNPs and DOX. Furthermore, AgNPs size influences their interactions with DOX, as evidenced by differences in the heteroaggregates formation observed in biophysical experiments and further supported by in vitro biological assays. We detected reduction of tumor cell viability and/or colony sizes of the analyzed cancer cell lines, registering differences linked to the observed phenomenon. However, the effects may be limited to the outer borders of the tumor microenvironment as evidenced by the 3D model. Summing up, we observed diverse patterns of interactions and biological effects for different sizes of AgNPs with DOX providing insight how the nanoparticles’ size affects their interactions with other biologically active compounds. Moreover, obtained data can be further used in experiments on the reduction of tumor size i.e. before the surgical intervention.
This article covers the topic of the tourist attractiveness of Tokyo in the opinion of 369 tourists from Poland. A definition of tourist attractiveness and its factors is provided. Basic information ...on Tokyo, including accommodation, eating facilities, and tourist traffic in the city is offered. The results of the research performed with the use of a survey questionnaire are demonstrated. The research shows that tourists’ most appreciated elements of Tokyo’s tourist attractiveness are transport accessibility, eating facilities, and cultural assets. The article’s hypotheses, that the most attractive seasons in Tokyo, tourism-wise, are spring and autumn, and that the most attractive monument in Tokyo is the oldest Buddhist temple, Sensō-ji, located in the Taitō district, were confirmed to be positive. The third hypothesis was also positively verified. The research shows that younger people positively assessed Tokyo as an attractive and friendly city more than older people.
The present study aimed to synthesize novel polycationic polymers composed of
-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and ...DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)
-O2Oc-NH
, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-Dap(GO2)
-O2Oc-NH
and typical for large proteins.
Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the ...structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.
Lsr2 is a nucleoid-associated protein (NAP) that has been found strictly in actinobacteria, including mycobacteria. It is a functional homolog of histone-like nucleoid-structuring protein (H-NS); it ...acts as a DNA-bridging protein that plays a role in chromosomal organization and transcriptional regulation. To date, the studies on Lsr2 have focused mainly on
In this study, we analyze the role of Lsr2 as a transcription factor in
, a saprophytic bacterium whose natural habitat (soil and water) substantially differs from those of the obligatory mycobacterial pathogens. Our chromatin immunoprecipitation-sequencing (ChIP-seq) data revealed that Lsr2 binds preferentially to AT-rich regions of the
chromosome. We found that Lsr2 acts mainly as a repressor, controlling gene expression either directly by binding promoter regions or indirectly through DNA loop formation and DNA coating. One of the Lsr2-repressed genes encodes polyketide synthase (MSMEG_4727), which is involved in the synthesis of lipooligosaccharides (LOSs). An
strain deprived of Lsr2 produces more LOSs, which is mirrored by changes in the smoothness of cells and their susceptibilities to antibiotics. Unlike
,
additionally encodes a paralogue of Lsr2, MSMEG_1060, which is a novel member of the mycobacterial NAP family. The Lsr2 and MSMEG_1060 proteins exhibit different DNA binding specificities and chromosomal localizations. Our results suggest that these proteins help
cells cope with stress conditions, including hypoxia and exposure to antibiotics. Thus, the present work provides novel insight into the role of Lsr2 paralogues in the ability of a saprophytic mycobacterial species to adjust to environmental changes.
Nucleoid-associated proteins (NAPs) are the most abundant proteins involved in bacterial chromosome organization and global transcription regulation. The mycobacterial NAP family includes many diverse proteins; some are unique to actinobacteria, and many are crucial for survival under stress (e.g., HupB and Lsr2) and/or optimal growth conditions (e.g., mycobacterial integration host factor mIHF). Here, we present a comprehensive study concerning two functional homologues of mycobacterial H-NS: Lsr2 and its paralogue from
, MSMEG_1060. We found that Lsr2 plays a role in transcriptional regulation, mainly by repressing gene expression via DNA loop formation and/or DNA-coating mechanisms. Intriguingly, the number of Lsr2-mediated genes was found to increase under hypoxia. Compared to Lsr2, MSMEG_1060 exhibits a different DNA binding specificity and chromosomal localization. Since tuberculosis remains a serious worldwide health problem, studies on stress response-mediating agents, such as Lsr2, may contribute to the development of novel antituberculosis drugs.
Indomethacin (IMC) and quercetin (QUE) as typical models of anti-inflammatory drugs were loaded into the micelles of new amphiphilic graft copolymers, comprising caprolactone 2-(methacryloyloxy)ethyl ...ester (CLMA) units in the main chain and poly(meth)acrylic acid side chains (PAA/PMAA), which were studied as the carriers of drugs. The macromolecules were self-assembled by solvent evaporation or dialysis. The critical micelle concentration (CMC) ranged from 0.015 to 0.199mg/ml. The copolymer composition, grafting degree and length of side chains, nature and content of hydrophobic/hydrophilic part, were investigated as the main parameters responsible for the properties of nanoparticles including their stability, core-drug interactions, improved drug solubility, and in consequence the efficiency of drug-loading and drug release profiles. The hydrodynamic diameters of particles measured by dynamic light scattering (DLS) ranged from 50 to 275nm, and increased after loading with drug. In vitro release experiments performed at various pH (5.0 and 7.4) indicated faster release behavior from nanoparticles in acidic conditions (55–95% vs. 25–45% within 75h).
PDF
