[Endotoxin production in the early stages of urgent abdominal pathology and correction ways]. / Endogennaya intoksikatsiya v rannie sroki pri urgentnoi patologii organov bryushnoi polosti i puti ee korrektsii.

OBJECTIVE: To study the features of the development of endogenous intoxication in association with damage to the organs of the detoxification system, the severity of catabolic processes associated with the activity of peroxidation of membrane lipids and phospholipases in various urgent pathology of the abdomen, on the basis of which to develop a new pathogenetically based approach to the prevention of the progression of surgical endotoxicosis. MATERIAL AND METHODS: Clinical section. The studies were conducted in 162 patients with acute urgent pathology (seven groups) with predominant aseptic inflammation (acute severe pancreatitis, acute intestinal obstruction, acute catarrhal cholecystitis) and pronounced purulent-necrotic phenomena (acute peritonitis, acute destructive cholecystitis with empyema of the gallbladder). The severity of endogenous intoxication, the functional state of detoxification system organs (liver, kidneys and intestines), the activity of peroxidation of membrane lipids and phospholipases were evaluated in dynamics. In the last two groups of patients with pancreatitis and peritonitis, complex therapy included remaxol. Experimental section. The experiments were conducted on mongrel dogs that were modeled with acute peritonitis (n=15) or biliary pancreatitis (n=15). A complex of studies similar to those in the clinic was applied in dynamics, but in the tissue structures of the liver, kidneys and intestines, the activity of peroxidation of membrane lipids and phospholipases, as well as the phospholipid composition, was studied. RESULTS: Experimental and clinical studies have shown that in the studied diseases with a different nature of the inflammatory process (aseptic or purulent) in the initial periods, the production of toxins is largely associated with catabolic phenomena associated with a significant activation of peroxidation of membrane lipids and phospholipases. A noticeable increase in the activity of these processes was noted regardless of the nature and severity of the inflammatory process and is associated with the severity of endogenous intoxication and the dysfunctional state of the detoxification system organs. Subsequently, there is a decrease in the detoxification ability of the body against the background of stabilization or even reduction of catabolic phenomena. It is shown that a significant increase in the activity of trigger agents of membrane-stabilizing phenomena occurs regardless of the nature and severity of the inflammatory process. Their significant activity was experimentally established in the tissue structures of the detoxification system organs themselves, which caused the development of pronounced membrane-stabilizing phenomena. It has been shown that the use of remaxol in the complex therapy of patients leads to a noticeable decrease in the activity of membrane modulating agents, which leads to a significant decrease in the severity of endogenous inetoxification syndrome, regardless of the nature of inflammation. This fact was an additional evidence base for the conceptual rethinking of the significance of catabolic phenomena in the pathogenesis of endotoxin production in the early stages of the studied diseases. CONCLUSION: Experimental and clinical grounds have been obtained for a conceptual rethinking of the role of membrane-stabilizing agents in flooding the body with toxins at the beginning of the disease. One of the leading roles of peroxidation of membrane lipids and phospholipase systems in catabolic processes in the earliest periods of urgent abdominal diseases and in the defeat of detoxification system organs in the subsequent ones is shown.

[Advanced regenerative rehabilitation technologies. Successful use of autologous adipose tissue in a patient with cutaneous tracheal fistula]. / Sovremennye tekhnologii regenerativnoi reabilitatsii patsientov. Opyt uspeshnogo ispol'zovaniya autologichnoi zhirovoi tkani u patsienta s kozhno-trakheal'nym svishchom.

According to the published data, the rate of fistula formation in the postoperative period ranges from 0.3% to 29%. Currently, due to the high risk of suture failure in the postoperative period and tracheal and esophageal fistulas formation, the options for fistulas treatment by the regenerative rehabilitation methods are under consideration. The frequency of fistula healing and reduction of scar deformities after adipose tissue autografting ranges from 43% to 55%. OBJECTIVE: To develop the minimally invasive technology for postoperative fistulas treatment and describe the real-world experience of its use in terms of hospital stay reduction and patients' quality of life improvement. MATERIAL AND METHODS: Patients were selected as part of a pilot research project (Development of Personalized Approaches to Regenerative Rehabilitation for Surgical Patients, # 0394-2020-0011). Patients' quality of life was assessed using the Russian version of the EQ-5D-5L questionnaire (Russian version for Russia). This article presents a clinical example of successful repair of a cutaneous tracheal fistula by autografting of adipose tissue three months after fistula formation. RESULTS: In one month after autografting of adipose tissue, the tracheobronchial secretion through the fistula has stopped. According to the follow-up computed tomography of the neck and tracheobronchoscopy, the fistulous passage is not observed. CONCLUSION: The presence of a fistula worsens the patient's quality of life and increases the risk of secondary infection. When treating patients with tracheal fistulas of high and extremely high surgical risk, the method of autografting of adipose tissue can be a safe and effective alternative to conventional surgery. Further study of this method of regenerative rehabilitation is required.

[Sjogren's disease/syndrome in combination with autoimmune liver damage. 20-years observation]. / Bolezn'/sindrom Shegrena v sochetanii s autoimmunnym porazheniem pecheni. 20-letnee nablyudenie.

Sjogren's disease (BS) is a systemic autoimmune disease, the main symptoms of which are associated with dry eyes, mouth, skin, respiratory and gastrointestinal tract, etc., but there are a large number of extra-vascular symptoms that can accompany this disease: weakness, swelling of the lymph nodes, respiratory failure, pain and swelling of the joints, rash, dysphagia, gastro-esophageal reflux, lymphoma, etc. In addition, the etiology of this disease remains unknown, and the pathogenesis is partially studied. The diagnosis of Sjogren's disease is complicated by a variety of clinical manifestations, as well as subacute and chronic variants of the course of the disease, in addition, the diagnostic criteria are periodically changed. It is recommended to adhere to the domestic diagnostic criteria considering the presence of laboratory signs of an autoimmune disease. The article presents a clinical observation of a patient with the development of the main symptoms and signs of BS and autoimmune hepatitis for 20 years. Recurrent mumps or chronic bilateral enlargement of the parotid salivary glands may be the first symptom of Sjogren's disease, which is recommended to extend the examination in order to identify other, sometimes latent signs of the disease.

[Giant Aneurysms in Coronary Arteries of a Young Woman].

Coronary arteries aneurysms with their thrombotic occlusion are known to be detected in young patients who have suffered Kawasaki disease in childhood. The other vascular beds are usually not involved. In the literature one can find not enough information regarding diagnostics of this pathology, as well as no specific treatment algorithm. We present here a clinical case of re-emergence of giant aneurysms of coronary arteries in the young female patient with subsequent immuno-histological confirmation of previous Kawasaki disease.

[Differentiation of tortoises of the genera Testudo and Agrionemys (Testudinidae) based on the polymorphism of nuclear and mitochondrial markers].

Based on polymorphism of the 12S rRNA gene and RAPD markers, differentiation of 122 tortoise individuals belonging to the three species of genus Testudo (T. kleinmanni, T. marginata, and T. graeca), six subspecies of T. graeca (T. g. nikolskii, T. g. pallasi, T. g. armeniaca, T. g. zarudnyi, T. g. terrestris, T. g. ibera), and two subspecies of the Central Asian tortoise Agrionenemys horsfieldii (A. h. horsfieldii, A. h. kazakhstanica) was performed. For comparison, 32 known sequences of 12S rRNA gene (392 bp) from tortoises of the two genera inhabiting the territories of Europe, Asia, and Africa were used. In the populations of A. horsfieldii, a total of six haplotypes; including three newly described variants, were identified. In the examined tortoises of the genus Testudo, eleven 12S rRNA haplotypes were identified. One new haplotype was detected in T. kleinmanni. Among the eight subspecies of T. graeca, eight haplotypes were identified, with four newly described ones. The reported RAPD markers generally supported the reconstructions obtained with the use of the mitochondrial marker. Similarly to the 12S rRNA-based reconstructions, two independent clusters included representatives of the two genera, Agrionemys and Testudio. Among the latter, representatives of T. marginata and T. kleinmanni, as well as T. graeca, with high statistical support values, formed two reciprocally monophyletic groups. Compared to the mitochondrial markers, RAPDs more statisticallysignificantly discriminated the sample of T. g. terrestris and the four subspecies, T. g. ibera, T. g. armeniaca, T. g. pallasi, and T. g. nikolskii. In almost all cases except the representatives of T. g. ibera, the representatives of each of four subspecies formed individual subclusters. The geographical haplotype distribution patterns and possible evolutionary scenario of the origin and dispersal of tortoises of the two genera are discussed.

Topology of mRNA chain in isolated eukaryotic double-row polyribosomes.

In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The "long" (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular ("collapsed rings"), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3'-end) and initiating 40S subunits (5'-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.

Leader sequences of eukaryotic mRNA can be simultaneously bound to initiating 80S ribosome and 40S ribosomal subunit.

Binding of mRNA leader sequences to ribosomes was studied in conditions of a cell-free translation system based on wheat germ extract. Leader sequence of TMV mRNA (the so-called omega-RNA sequence) was able to bind simultaneously 80S ribosome and 40S ribosomal subunit. It was found that nucleotide substitutions in omega-RNA resulting in destabilization of RNA structure have no effect on the complex formation with both 80S ribosome and 40S ribosomal subunit. Leader sequence of globin mRNA is also able to form a similar joint complex. It is supposed that the ability of mRNA leader sequences to bind simultaneously 80S ribosome and 40S subunit is independent of leader nature and may reflect previously unknown eukaryotic mechanisms of translation initiation.

Functional activity of neutrophils in diabetes mellitus and coronary heart disease: role of myeloperoxidase in the development of oxidative stress.

We performed a comparative analysis of functional activity of neutrophils in patients with type 2 diabetes mellitus with and without symptoms of CHD. Enhanced H2O2 production by neutrophils in response to N-formyl-Met-Leu-Phe (fMLP) was found in patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus associated with CHD, fMLP-induced release of myeloperoxidase from azurophilic granules of neutrophils was reduced and plasma myeloperoxidase level was elevated. Increased peroxidase activity of myeloperoxidase, reduced plasma catalase activity, and increased levels of TBA-reactive lipid peroxidation products and oxidized glutathione were detected in patients of both groups. Since myeloperoxidase is an important neutrophilic mediator of oxidative stress, its increased activity in the blood can be an additional marker of oxidative stress and cardiovascular risk in patients with diabetes mellitus.

[Characteristic of extracellular lectin in Bacillus subtilis IMV B-7014].

The monosaccharide and fatty acid composition of water and organic phase of lectin was studied. It was established that extracellular lectin contains 15.0% of protein, 4.6% of carbohydrates, 1.0% of nucleic acids and 16.14% of lipids. The following monosaccharides were presented in the lectins: mannose, ribose, glucose and ramnose. It was established that both native lectin and its lipophylic fraction were composed of fatty acids from C15 to C19. During aggressive methanolysis anti-iso-C15 quantity was increased with a synchronous decrease of the content of all octadecanoic acid types.

Apomyoglobin mutants with single point mutations at val10 can form amyloid structures at permissive temperature.

Formation of amyloid-like protein aggregates in human organs and tissues underlies many serious diseases, therefore being in the focus of numerous biochemical, medical, and molecular biological studies. So far, formation of amyloids by globular proteins has been studied mostly under conditions that strongly destabilized their native structure. Here we present our results obtained at permissive temperature by thioflavin T fluorescence, far UV CD, IR spectroscopy, and electron microscopy. We used apomyoglobin and its mutants with Ala or Phe substituted for Val10 that are structurally close to wild type apomyoglobin. It is shown that at permissive temperature the ability of the protein to form amyloids depends on the extent of its structural destabilization, but not on hydrophobicity of the substituting residue. A possible difference between amyloids formed by strongly destabilized proteins and those yielded by proteins with a slightly fluctuating native structure, as well as the stroke and infarction effect on the ability of proteins to form amyloid structures, are discussed.

Effect of ion doping with donor and acceptor impurities on intensity and lifetime of photoluminescence from SiO2 films with silicon quantum dots.

Doping with donor and acceptor impurities is an effective way to control light emission originated from quantum-size effect in Si nanocrystals. Combined measurements of photoluminescence intensity and kinetics give valuable information on mechanisms of the doping influence. Phosphorus, boron, and nitrogen were introduced by ion implantation into Si+ -implanted thermal SiO2 films either before or after synthesis of Si nanocrystals performed at Si excess of about 10 at.% and annealing temperatures of 1000 and 1100 degrees C. After the implantation of the impurity ions the samples were finally annealed at 1000 degrees C. It is found that, independently of ion kind, the ion irradiation (the first stage of the doping process) completely quenches the photoluminescence related to Si nanocrystals (peak at around 750 nm) and modifies visible luminescence of oxygen-deficient centers in the oxide matrix. The doping with phosphorus increases significantly intensity of the 750 nm photoluminescence excited by a pulse 337 nm laser for the annealing temperature of 1000 degrees C, while introduction of boron and nitrogen atoms reduces this emission for all the regimes used. In general, the effective lifetimes (ranging from 4 to 40 micros) of the 750 nm photoluminescence correlate with the photoluminescence intensity. Several factors such as radiation damage, influence of impurities on the nanocrystals formation, carrier-impurity interaction are discussed. The photoluminescence decay is dominated by the non-radiative processes due to formation or passivation of dangling bonds, whereas the intensity of photoluminescence (for excitation pulses much shorter than the photoluminescence decay) is mainly determined by the radiative lifetime. The influence of phosphorus doping on radiative recombination in Si quantum dots is analyzed theoretically.

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila.

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). We have also measured the levels of 20E in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). We have studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis and demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed.

Amyloidogenic and anti-amyloidogenic properties of recombinant transthyretin variants.

Most transthyretin (TTR) mutations lead to TTR amyloid depositions in patients with familial amyloidotic polyneuropathy and familial amyloidotic cardiomyopathy. However, though an amyloidogenic protein itself, TTR inhibits aggregation of Alzheimer's amyloid beta protein (A beta) in vitro and in vivo. The pathogenic relationship between two amyloidogenic processes remains unclear. To understand how TTR mutations influence the ability of TTR to inhibit A beta amyloidosis, forty-seven recombinant TTR variants were produced and analyzed. We showed that all recombinant proteins formed tetramers and were functional in thyroxine binding. Acid denaturation at pH 3.8 resulted in aggregation and fibril formation of all TTR variants. However, only TTR G42 and TTR P55 formed fibrils at pH 6.8. Most TTR variants bound to A beta and inhibited A beta aggregation in vitro. TTR variants S64, A71, Q89, V107, H114 and I122 revealed decreased binding to A beta and decreased inhibition of A beta aggregation. Only TTR G42 and TTR P55 completely failed to bind A beta and to inhibit A beta aggregation. We suggest that TTR variants characterized by decreased binding to A beta or by decreased inhibition of A beta aggregation in vitro may contribute to A beta amyloid formation in vivo. These TTR variants might be important targets for epidemiological studies in Alzheimer's disease.

Shift of fibril-forming ability of the designed alpha-helical coiled-coil peptides into the physiological pH region.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.

Structure of a beheaded 30 S ribosomal subunit from Thermus thermophilus.

The 22 S ribonucleoproten particles containing the 5' (body) and the central (platform) domains of the Thermus thermophilus 30 S subunit has been studied by sedimentation, neutron scattering and electron microscopy. The RNP particles have been obtained by oligonucleotide-directed cleavage of 16 S RNA with ribonulease H in the region of the 900th nucleotide of the protein-deficient derivatives of the 30 S subunits. It is shown that these RNP particles are very compact, though their form and dimensions differ slightly from those expected from the electron microscopy model of the 30 S subunit beheaded by computer simulation. The particles are subdivided into two structural domains whose mutual arrangement differs from that of the corresponding morphological parts of the native 30 S subunit. Electron microscopy demonstrates that the mutual arrangement of domains in the RNP particles is not strictly fixed suggesting that interaction with the third domain of the 30 S subunit is a requisite for their correct fitting.

In vitro assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit.

A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.

Simulation of constituent processes of anaerobic degradation of organic matter by the "methane" model.

The model of anaerobic digestion described earlier by the authors was used for analysis of the different phases of the process. It was shown that at the glucose conversion a coexistence of hydrogen-producing acidogenic bacteria and hydrogen-utilizing non-methanogenic bacteria causes a hydrogen partial pressure decrease at an increase of solids retention time (i), the intensity of the negative feed-back effect in sulfate-reduction through hydrogen sulfide formation is regulated by the pH level during an oscillation dynamics in acetate/sulfate system (ii), under the toxicity influence the processes of methanogenesis and acetogenesis together with hydrolysis may be rate-limiting steps in the anaerobic system with particulate substrate degradation (iii).

Thermus thermophilus ribosomes for crystallographic studies.

Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies.