Luminescent probe method in the study of the interaction of glycated human serum albuminwith non-glycated human serum albumin

Background and Objectives: The development and functioning of all living beings ends with the inevitable aging process, as a result of which the activity of all organs and the body as a whole is suppressed, which leads to imminent death. Protein glycation is considered to be one of the causes of aging. This process takes place throughout life, but it intensifies with age. Protein glycation is a reaction of covalent coupling of free amino groups of proteins and reducing carbohydrates, which proceeds without the participation of enzymes and leads to disruption of protein functions. This process is unregulated, as it occurs without the participation of biological catalysts. As a result of glycation of proteins in humans, inflammatory processes occur in the body and a number of diseases such as heart attack, stroke, atherosclerosis, cataract, glycemia, Alzheimer’s disease, diabetes mellitus, etc. develop. In the tasks of medical diagnostics, methods of monitoring the state of proteins in the human body are necessary. In this regard, the work is devoted to the study of the processes of interaction of human serum albumin globules (HSA) with globules of human glycated serum albumin (gHSA). Materials and Methods: In conducting a study of the spectral-kinetic characteristics of the eosin luminescent probe in solutions of glycated and non-glycated HSA, as well as in a mixture of glycated and non–glycated HSA, an exponential dependence of the second order was used to approximate the dependencies of DF (delayed fluorescence) and PHOS (phosphorescence), and an anisotropy equation was used to assume the formation of the gHSA-HSA complex. Results: It has been found that the intensity and kinetics of quenching of delayed fluorescence and phosphorescence of the eosin fluorescent probe associated with proteins are sensitive to the ratio of glycated and non-glycated proteins in solution. To explain the increase in the intensity and lifetime of eosin phosphorescence during the transition from a solution of HSA to a mixture of HSA and gHSA, it is assumed that the globules of HSA and gHSA form a complex of the composition of gHSA-HSA, as a result of diffusion encounters. The rotational mobility of this complex is much less than the separate globules of HSA and gHSA. The formation of the complex is confirmed by an increase in the anisotropy of delayed fluorescence and phosphorescence of eosin in a mixture of HSA and gHSA. Conclusion: The obtained results of the work can be used to diagnose the presence of a complex of glycated with non-glycated proteins in human blood plasma.&nbsp

S-layer protein 2 of 'Lactobacillus crispatus' 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis

Interglobular Diffusion of an Energy Donor in Triplet-Triplet Energy Transfer in Proteins

The triplet-triplet energy transfer between polar molecules of luminescent probe (eosin) as an energy donor and nonpolar molecules of energy acceptor (anthracene) is studied. Both the donor and the acceptor are bound to human serum albumin by noncovalent bonds. A dependence of rate constant of triplet-triplet energy transfer on human serum albumin concentration is revealed. A rate constant of eosin output from protein globules is determined. It is shown that the energy transfer occurs as a result of interglobular diffusion of eosin. The obtained results indicate that a protein-luminescent probe based sensor can be used for testing a concentration of polycyclic aromatic hydrocarbons in proteins

Toward a Selective Analysis of Heavy Metal Salts in Aqueous Media with a Fluorescent Probe Array

Detection of heavy meals in aqueous media challenges worldwide research in developing particularly fast and affordable methods. Fluorescent sensors look to be an appropriate instrument for such a task, as recently they have been found to have made large progress in the detection of chemical analytes, primarily in the environment, along with biological fluids, which still suffer from not enough selectivity. In this work, we propose a new fluorescent method to selectively recognize heavy metals in an aqueous solution via employing an array of several fluorescent probes: acridine yellow, eosin, and methylene blue, which were taken as examples, being sensitive to a microsurrounding of the probe molecules. The exemplary sensor array generated six channels of spectral information through the use of various combinations of excitation and detection wavelengths. Following the known multisensor approach, we applied a linear discriminant analysis to selectively distinguish the vector signals from the sensor array from salts of heavy metals—Cu, Pb, Zn, Cd, and Cz—at the concentration ranges of 2.41 × 10−6–1.07 × 10−5 M, 2.8 × 10−5–5.87 × 10−4 M, 1.46 × 10−6–6.46 × 10−6 M, 1.17 × 10−8–5.2 × 10−8 M, and 2.11 × 10−6–9.33 × 10−6 M, respectively. The suggested approach was found to be promising due to it employing only one cuvette containing the test solution, simplifying a sample preparation when compared to preparing a variety of solutions in tests with single fluorescence probes

Infrared spectra of organic liquids and cluster model of substance

We consider the effective field theory based on the consideration of the rotation and libration of molecules in surroundings represented as effective clusters. The suitable distribution function with respect to the number of particles, which form a cluster, is discussed. This approach is applied to the forecasting of infrared spectrum frequencies of liquids using liquefied inert gases, nitrogen, oxygen, benzene and water as examples

S-layer protein 2 of vaginal 'Lactobacillus crispatus' 2029 enhances growth, differentiation, VEGF production and barrier functions in intestinal epithelial cell line Caco-2

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes

\u3cem\u3e Limosilactobacillus Fermentum\u3c/em\u3e 3872 That Produces Class III Bacteriocin Forms Co-aggregates with the Antibiotic-resistant\u3cem\u3e Staphylococcus Aureus\u3c/em\u3e Strains and Induces Their Lethal Damage

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals