Interactions of intercellular adhesion molecules of the selectin family with glycoconjugates of cell membranes mediate the initial stage of the adhesion cascade, which recruits leukocytes circulating in the bloodstream to sites of infection or damage. The formation of heterotypic cell aggregates between individual cells of hematopoietic and non-hematopoietic origin may be involved in processes leading to inflammation, thrombosis, and metastasis. A key protein, the dimeric glycoprotein PSGL-1, a P-selectin glycoprotein ligand, plays an important role in the binding of selectins, serving as a ligand for all three selectins. PSGL-1 combines signals activating various biochemical pathways during binding and rolling of leukocytes. The integration of these signals leads to activation of leukocytes, integrin-mediated arrest, restructuring of the cyto- skeleton of interacting cells, polarization, and subsequent diapedesis of leukocytes into surrounding tissues. The multilevel effect of PSGL-1 on cellular traffic in the physiological and inflammatory states is largely determined by posttranslational modifications, among which an important place is given to specific O- and N-glycosylation and sulfation. In this review, we discuss modifications of PSGL-1 associated with the initiation of biochemical pathways, as well as its interactions, which make it possible to classify this molecule as signaling, paying special attention to the mechanisms leading to pathology, including cardiovascular.
The substrate properties of six pairs of fluorescently labeled deoxyuridine and deoxycytidine triphosphates (Cy5-dUTPs and Cy5-dCTPs) in PCR with Taq polymerase were compared. In each pair, the modified dU and dC contained identical fluorescently labeled Cy5 substituents; for different pairs, the sub-stituent structures differed in the length of the linker between the nitrogenous base and the fluorophore, the length of the linker between the quaternary ammonium group and the second heterocycle of the fluorophore, as well as the structure of the fluorophore itself. DNA fragments of Staphylococcus aureus (AT-rich template) and Mycobacterium tuberculosis (GC-rich) were used as templates. With both templates, deoxycytidine derivatives showed slightly higher amplification efficiency (E). The influence of the fluorophore structure and the GC-composition of the template on the kinetics of the reaction was insignificant. At the same time, a high incorporation efficiency was observed on the AT-rich template for uridine derivatives, and on the GC-rich template for cytidine derivatives (and in both cases, for substituents with a longer linker length). Nevertheless, the specific incorporation density, which takes into account the number of similar nucleotides in the DNA chain, was in all cases higher for dU derivatives. It was found that in pairs with similar fluorophore modifications, uridine derivatives, compared with cytidine, are characterized by a higher incorporation density, regardless of the composition of the template, but at the same time they have a greater inhibitory effect. The results obtained will increase the sensitivity of fluorescence analysis using the immobilized phase (microarray analysis).
The COVID-19 pandemic has triggered the development of new directions in vaccine development, among which DNA- and mRNA-based technologies are particularly noteworthy. The platform based on DNA vaccines is developing particularly intensively due to their high stability at ambient temperature and the ability to activate both humoral and cellular immunity. The full cycle of DNA vaccine creation, which includes the construction of plasmid DNA, obtaining a producer strain, fermentation, and purification, takes 2-4 weeks. In addition, the production technology of such vaccines does not require working with dangerous pathogens, which significantly simplifies the process of their production and reduces the overall cost. Over more than 30 years of rapid development, DNA vaccine technology continues to undergo changes. Currently, there is a licensed DNA vaccine for the prevention of COVID-19, and many candidate prophylactic vaccines against viral and bacterial diseases are in clinical trials. This review covers not only the principles of constructing plasmid DNA vaccines, but also new technologies for obtaining DNA constructs, such as minicircular DNA, MIDGE DNA, and Doggybone^(™) DNA. New types of DNA vaccines are interesting because they consist only of the most essential elements for activating the immune response. Such constructs completely lack the sequences necessary for the production of plasmid DNA in bacterial cells, for example, the antibiotic resistance gene. One of the key problems in the development of a DNA vaccine is the method of its delivery to target cells. Currently, various delivery methods are used, both chemical and physical, which are rapidly developing and have already proven themselves to be reliable and effective. The characteristics of some of the most promising methods are also presented in this review.
Studies of antibacterial secondary metabolites of marine micromycete fungi as an element of a modern strategy for the search for new antibiotics are considered. More than half of the drugs currently used in practice have been isolated from bacteria (Bacteria) and actinomycetes (Actinomycetes); however, the first antimicrobial compounds were isolated from mycelial fungi (Ascomycetes), and it is obvious that their potential has not been exhausted. Marine fungi occupy a separate niche due to the peculiarities of their habitats, which also affect their production of low molecular weight compounds. This paper provides information on the secondary metabolites of marine fungi acting against those bacterial targets focused by the modern search for new antibiotics and discusses a strategy for investigating the antibacterial activity of marine fungal metabolites.
Thiocyanate dehydrogenase is enzyme catalyzing transformation of a thiocyanate ion into a cyanate ion with outcome of two electrons, two protons and a neutral atom of sulphur. Earlier structures of thiocyanate dehydrogenase from Thioalkalivibrio paradoxus were solved. Despite not perfect quality of the structures (twinning and pronounced anisotropy of the crystals, incomplete occupancy of the copper ions, absence of data for complexes with analogues of the substrate), there was suggested a mechanism of the enzyme functioning based on those structures. Recently at atomic resolution there have been solved structures of a gene-modified copy of relative enzyme from Ptlomicrobium methylotrophicum for free protein and its complex with thiourea. In the new structures copper ions of the active site possess complete occupancy. In these structures it is possible to reliably identify two conformations of the protein molecule with opened and closed active sites. The new structural high resolution data also allowed us to determine the presence of the superposition of different states of the copper ions for each of the two conformations. In each state the copper ions have different oxidation degrees, different corresponding ligands and partial occupancies. The ion charges were determined according to the ions coordination. In the protein molecule with the closed active site the complexes with inhibitor (thiourea ion) and molecular oxygen are observed. The complex with thiourea allows us to model binding of thiocyanate ion to the enzyme molecule. Taking into account the changes of the structures in the opened and closed conformations, a mechanism of the attacking oxygen ligand activation is suggested. A new scheme of the enzymatic reaction is discussed.
The kinetics of amplification and the features of individual and simultaneous incorporation of modified deoxynucleoside triphosphates in DNA during rolling circle amplification (RCA) have been studied. This study was carried out for six pairs of Sy5-labeled triphosphates of deoxyuridine (dU) and deoxycytidine (dC) previously synthesized with similar fluorescent substituents inside the pair. The effect of the linker length between the fluorophore and the pyrimidine base on the incorporation density was determined: nucleotides with a linker length of six carbon atoms are embedded in a growing DNA chain better than with three carbon atoms. It was found that the combined introduction of triphosphates into the reaction in an equivalent total concentration does not enhance the inhibitory effect, which gives grounds for a more detailed study of the simultaneous use of labeled dU and dC.
Long-term pharmacotherapy in patients with schizophrenia can provoke antipsychotic-induced obesity. This side effect does not always meet the criteria for metabolic syndrome (MS), primarily central obesity. However, this significantly reduces the quality of life of patients and is a risk factor for the development of many diseases. In humans, the NOS1AP gene product is involved in adipogenesis, dendrite maturation, mnemonic processes, and impulse transmission by NMDA receptors. We hypothesized that NOS1AP gene polymorphisms are associated with metabolic parameters in patients with schizophrenia. We examined 491 patients of Slavic nationalities with an established diagnosis of schizophrenia. All participants underwent anthropometric examination to determine waist circumference and the total and visceral fat content using bioimpedance analysis and caliperometry. The biochemical parameters of the blood serum were evaluated by standard methods. MS components were determined according to the International Diabetes Federation criteria. DNA was isolated from peripheral blood leukocytes by the standard phenol-chloroform method. Three SNPs in the NOS1AP gene were selected for genotyping. The alleles of the studied polymorphisms were determined by real-time PCR. As a result, statistically significant differences in the groups of patients with different levels of visceral fat in the distribution of the allele frequency of the s12143842 NOS1AP polymorphism, as well as differences in the levels of visceral fat depending on the rs10494366 NOS1AP genotype were revealed. For the first time, an association of NOS1AP gene polymorphisms with the formation of visceral fat levels in patients with schizophrenia was established. The results obtained can be further used to develop genetic panels to predict the development of adverse metabolic effects during antipsychotic therapy for schizophrenia.
Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by progressive motor impairment due to the death of dopaminergic neurons in the substantia nigra (SN) of the brain. PD affects more than 1% of the population over 60 years of age worldwide. Despite significant progress in understanding the pathogenesis of PD, including genetic and biochemical aspects, current therapy is limited to symptomatic treatments. Recent evidence suggests that impaired autophagy leads to the accumulation of abnormal proteins and, particularly, α-synuclein, aggregated forms of which are neurotoxic to dopaminergic neurons in the SN. Notably, PD is predominantly sporadic. However, monogenic PD forms have also been described. PD forms associated with mutations of the GBA1 or LRRK2 gene are among the most common PD forms with known etiology. Leucine-rich repeat kinase 2 (LRRK2), which is encoded by LRRK2, and the lysosomal enzyme glucocerebrosidase (GCase), which is encoded by GBA1, are involved in the same endolysosomal pathway. LRRK2 and GCase dysfunction reported in PD, especially in cases with mutations of the respective genes, can impair the endolysosomal pathway, the lysosomal function, and possibly autophagy. The review highlights the molecular mechanisms of autophagy and the prospects for targeted therapy of PD via induction of autophagy by influencing the key players in the process.
DNA methylation is one of the most important mechanisms closely involved in the epigenetic regulation of gene expression. However, the relationship between DNA methylation and expression is not completely understood. There are reported examples of changes in DNA methylation being the cause of gene expression, and vice versa - examples of changes in gene expression to entail changes in methylation. Earlier, we introduced the concept of CpG traffic lights - individual CpG sites methylation levels of which significantly correlate with the expression of a gene nearby - and showed their important role in the regulation of enhancers. In this work, we show that the levels of CpG traffic lights methylation are heterogeneous in the cell population, and suggest that this feature is caused by their dynamic demethylation. The 5hmC enrichment and TET2 localization sites in CpG traffic lights confirm our hypothesis. In order to find out whether methylation of CpG sites is the cause or the consequence of the gene expression, we applied the assessment of the causation direction method developed by Jonas Peters and co-authors. We determined that in CpG sites where changes in methylation are predicted to be the cause of gene expression changes (M→E CpG sites), methylation levels are more stable in different cells and active demethylation occurs less often than in CpG sites where presumably changes in gene expression cause changes in methylation (E→M CpG sites). We also show a greater share of M→E CpG sites in the promoter regions than in the bodies of the genes where E→M CpG sites are more common. Based on these observations, we believe that methylation of M→E CpG sites is stable and functions as an "on/off" switch. On the contrary methylation of E→M sites is dynamic, variable between cells in a cell population, and is caused primarily by active demethylation.
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