1461 research outputs found
Sort by
Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly
Get PDFThe binding of metal ions at the interface of protein complexes presents a unique and poorly
understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook
domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of
Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic
effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved
protein assembly. We have dissected the determinants of the stability contributed by the small
β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions
using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and
peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold
increase of the formation constant of the dimeric complex arises from favorable enthalpy
because of the increased acidity of the cysteine thiols in metal-free form and the structural
properties of the dimer. The dependence of the enthalpy on the domain fragment length is
partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy
compensation. This study facilitates understanding of the metal-mediated protein-protein
interactions in which the metal ion is critical for the tight association of protein subunits
Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology
Get PDFBackground
One of the major challenges limiting development of gene therapy is absence of efficient and safe gene carriers. Among the non-viral gene delivery methods, lipofection is considered as one of the most promising. In this study a set of cationic polyprenyl derivatives (trimethylpolyprenylammonium iodides – PTAI) with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles.
Methods
Optimization studies were conducted for PTAI in combination with co-lipid DOPE (dioleoylphosphatidylethanolamine) on DU145 human prostate cancer cells using: size and zeta potential measurements, confocal microscopy, fluorescein diacetate/ethidium bromide test, cell counting, time-lapse monitoring of cell movement, gap junctional intracellular coupling analysis, antimicrobial activity assay, red blood cell hemolysis test.
Results
The results show that the lipofecting activity of PTAI allows effective transfection of plasmid DNA complexes in negatively charged lipoplexes of 200 - 500 nm size into cells without significant side effects on cell physiology (viability, proliferation, morphology, migration and gap junctional intercellular coupling – GJIC). Moreover, PTAI-based vehicles exhibit a potent bactericidal activity against Staphylococcus aureus and Escherichia coli. The developed anionic lipoplexes are safe towards human red blood cell membranes which are not disrupted in their presence.
Conclusions
The developed carriers constitute a group of promising lipofecting agents of a new type and can be utilized as effective lipofecting agents in vitro and are a promising basis for in vivo applications
Arabidopsis SWI/SNF chromatin remodeling complex binds both promoters and terminators to regulate gene expression
Get PDFATP-dependent chromatin remodeling complexes
are important regulators of gene expression in Eukaryotes.
In plants, SWI/SNF-type complexes have
been shown critical for transcriptional control of
key developmental processes, growth and stress responses.
To gain insight into mechanisms underlying
these roles, we performed whole genome mapping
of the SWI/SNF catalytic subunit BRM in Arabidopsis
thaliana, combined with transcript profiling
experiments. Our data showthatBRM occupies thousands
of sites in Arabidopsis genome, most of which
located within or close to genes. Among identified direct
BRM transcriptional targets almost equal numbers
were up- and downregulated upon BRM depletion,
suggesting that BRM can act as both activator
and repressor of gene expression. Interestingly,
in addition to genes showing canonical pattern of
BRM enrichment near transcription start site, many
other genes showed a transcription termination sitecentred
BRM occupancy profile. We found that BRMbound
3� gene regions have promoter-like features,
including presence of TATA boxes and high H3K4me3
levels, and possess high antisense transcriptional
activity which is subjected to both activation and
repression by SWI/SNF complex. Our data suggest
that binding to gene terminators and controlling transcription
of non-coding RNAs is another way through
which SWI/SNF complex regulates expression of its
targets
Evidence of adaptation, niche separation and microevolution within the genus Polaromonas on Arctic and Antarctic glacial surfaces
Get PDFPolaromonas is one of the most abundant genera found on glacier surfaces, yet it’s ecology remains poorly described. Investigations made to date point towards a uniform distribution of Polaromonas phylotypes across the globe. We compared 43 Polaromonas isolates obtained from surfaces of Arctic and Antarctic glaciers to address this issue. 16S rRNA gene sequences, intergenic transcribed spacers (ITS) and metabolic fingerprinting showed great differences between hemispheres but also between neighboring glaciers. Phylogenetic distance between Arctic and Antarctic isolates indicated separate species. The Arctic group clustered similarly, when constructing dendrograms based on 16S rRNA gene and ITS sequences, as well as metabolic traits. The Antarctic strains, although almost identical considering 16S rRNA genes, diverged into 2 groups based on the ITS sequences and metabolic traits, suggesting recent niche separation. Certain phenotypic traits pointed towardscell adaptation to specific conditions on a particular glacier, like varying pH levels. Collected data suggest, that seeding of glacial surfaces with Polaromonas cells transported by various means, is of greater efficiency on local than global scales. Selection mechanisms present of glacial surfaces reduce the deposited Polaromonas diversity, causing subsequent adaptation to prevailing environmental conditions. Furthermore, interactions with other supraglacial microbiota, like algae cells may drive postselectional niche separation and microevolution within the Polaromonas genus
Physiological and Metagenomic Analyses of Microbial Mats Involved in Self-Purification of Mine Waters Contaminated with Heavy Metals.
Get PDFTwo microbial mats found inside two old (gold and uranium) mines in Zloty Stok and Kowary located in SW Poland seem to form a natural barrier that traps heavy metals leaking from dewatering systems. We performed complex physiological and metagenomic analyses to determine which microorganisms are the main driving agents responsible for self-purification of the mine waters and identify metabolic processes responsible for the observed features. SEM and energy dispersive X-ray microanalysis showed accumulation of heavy metals on the mat surface, whereas, sorption experiments showed that neither microbial mats were completely saturated with heavy metals present in the mine waters, indicating that they have a large potential to absorb significant quantities of metal. The metagenomic analysis revealed that Methylococcaceae and Methylophilaceae families were the most abundant in both communities, moreover, it strongly suggest that backbones of both mats were formed by filamentous bacteria, such as Leptothrix, Thiothrix, and Beggiatoa. The Kowary bacterial community was enriched with the Helicobacteraceae family, whereas the Zloty Stok community consist mainly of Sphingomonadaceae, Rhodobacteraceae, and Caulobacteraceae families. Functional (culture-based) and metagenome (sequence-based) analyses showed that bacteria involved in immobilization of heavy metals, rather than those engaged in mobilization, were the main driving force within the analyzed communities. In turn, a comparison of functional genes revealed that the biofilm formation and heavy metal resistance (HMR) functions are more desirable in microorganisms engaged in water purification than the ability to utilize heavy metals in the respiratory process (oxidation-reduction). These findings provide insight on the activity of bacteria leading, from biofilm formation to self-purification, of mine waters contaminated with heavy metals
Glycosaminoglycans and mucopolysaccharidosis type III
Get PDFMucopolysaccharidosis type III (MPS III), or Sanfilippo syndrome, is a lysosomal storage disease in which heparan sulfate is accumulated in lysosomes, as well as outside of cells, as the primary storage material. This disease is a complex of four conditions caused by dysfunctions of one of genes coding for lysosomal enzymes involved in degradation of heparan sulfate: SGSH (coding for heparan N-sulfatase) – causing MPS IIIA, NAGLU (coding for -N-acetylglucosaminidase) - causing MPS IIIB, HGSNAT (coding for acetyl CoA -glucosaminide acetyltransferase) - causing MPS IIIC), and GNS (coding for N-acetylglucosamine-6-sulfatase) – causing MPS IIID. The primary storage is responsible for some disease symptoms, but other arise as a result of secondary storage, including glycosphingolipids, and subsequent processes, like oxidative stress and neuroinflammation. Central nervous system is predominantly affected in all subtypes of MPS III. Heparan sulfate and its derivatives are the most commonly used biomarkers for diagnosis and prediction procedures. Currently, there is no therapy for Sanfilippo syndrome, however, clinical trials are ongoing for enzyme replacement therapy, gene therapy and substrate reduction therapy (particularly gene expression-targeted isoflavone therapy)
Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis
Get PDFMultiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite
various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive
T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here,
the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing
encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte
glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE)
– an animal model of MS.
Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming
units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological
analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were
measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE,
diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and
TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide
fragments could be an alternative strategy to induce oral tolerance for the treatment of MS
Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria.
Get PDFFactor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species
Apoptosis: its origin, history, maintenance and the medical implications for cancer and aging
Get PDFProgrammed cell death is a basic cellular mechanism. Apoptotic-like programmed cell death (called apoptosis in animals) occurs in both unicellular and multicellular eukaryotes, and some apoptotic mechanisms are observed in bacteria. Endosymbiosis between mitochondria and eukaryotic cells took place early in the eukaryotic evolution, and some of the apoptotic-like mechanisms of mitochondria that were retained after this event now serve as parts of the eukaryotic apoptotic machinery. Apoptotic mechanisms have several functions in unicellular organisms: they include kin-selected altruistic suicide that controls population size, sharing common goods, and responding to viral infection. Apoptotic factors also have non-apoptotic functions. Apoptosis is involved in the cellular aging of eukaryotes, including humans. In addition, apoptosis is a key part of the innate tumor-suppression mechanism. Several anticancer drugs induce apoptosis, because apoptotic mechanisms are inactivated during oncogenesis. Because of the ancient history of apoptosis, I hypothesize that there is a deep relationship between mitochondrial metabolism, its role in aerobic versus anaerobic respiration, and the connection between apoptosis and cancer. Whereas normal cells rely primarily on oxidative mitochondrial respiration, most cancer cells use anaerobic metabolism. According to the Warburg hypothesis, the remodeling of the metabolism is one of the processes that leads to cancer. Recent studies indicate that anaerobic, non-mitochondrial respiration is particularly active in embryonic cells, stem cells, and aggressive stem-like cancer cells. Mitochondrial respiration is particularly active during the pathological aging of human cells in neurodegenerative diseases. According to the reversed Warburg hypothesis formulated by Demetrius, pathological aging is induced by mitochondrial respiration. Here, I advance the hypothesis that the stimulation of mitochondrial metabolism leads to pathological aging
Type II and type V CRISPR effector nucleases from a structural biologist’s perspective
Get PDFThe type II and type V CRISPR effector nucleases Cas9 and Cpf1 are “universal” DNA endonucleases,
which can be programmed by an appropriate crRNA or sgRNA strand to cleave
almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In
this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context
in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been
obtained in the last two or three years that illustrate the mode of operation of these proteins. We
describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the
5’- or 3’-handles that help anchor the nucleic acid complexes to the proteins in a manner that is
independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and
Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called
protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix
(BH) promote the separation of target and non-target DNA strands and the formation of hybrids
between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding
of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very
uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key
developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases