[The forensic implications of the relationship between the proteolytic enzymes activity and the changes in NADH and FAD fluorescence intensity in skeletal muscle when determining the time of death (experimental study)]. / Sudebno-meditsinskoe znachenie vzaimosvyazi aktivnosti proteoliticheskikh fermentov i dinamiki intensivnosti fluorestsentsii kofermentov NADH i FAD v skeletnoi myshtse pri diagnostike davnosti nastupleniya smerti (eksperimental'noe issledovanie).

OBJECTIVE: Evaluation of the relationship between the activity of proteolytic enzymes (cathepsin D and calpains) and the dynamics of the fluorescence intensity of the coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the rats' skeletal muscles in relation to the time of death. The proteolytic activity of enzymes in rat skeletal muscle was determined at the postmortem time points corresponding to the most significant changes in the dynamics of coenzymes NADH and FAD fluorescence intensity. The proteolytic enzymes activity was found to be low during the period of increasing intensity of NADH fluorescence observed within 3 hours after death. An increase in the activity of proteolytic enzymes was registered in 4.5 hours after death which corresponds to the initial point of decrease in NADH fluorescence intensity. In 24 hours post-mortem, corresponding to increased FAD fluorescence intensity a significant decrease in the activity of calpains was found. The results of the study suggest that the nature of the postmortem dynamics of the fluorescence intensity of coenzymes is largely due to the peculiarities of intracellular proteolysis. The study results suggest that the pattern of post mortem changes in coenzyme fluorescence intensity is largely attributable to the specifics of intracellular proteolysis. The relationship between coenzyme fluorescence and molecular mechanisms of cell death confirms the viability and feasibility of laser-induced spectroscopy for post-mortem changes assessment when determining the time of death.

[Determination of the fluorescence intensity of coenzymes NADH and FAD in the skeletal muscle of the rat depending on the post-mortem interval]. / Opredelenie intensivnosti fluorestsentsii kofermentov NADN i FAD v skeletnoi myshtse krysy v zavisimosti ot davnosti nastupleniia smerti.

Aim of the study is to identify patterns of variations of the fluorescence intensity of NADH (reduced nicotinamide adenine dinucleotide) and FAD (oxidized flavin adenine dinucleotide) in the skeletal muscle depending on the time since death. For the evaluation of fluorescence intensity of the studied coenzymes, laser-induced spectroscopy in situ was used. We revealed the dynamic of the fluorescence intensity of NADH and FAD in the skeletal muscle of a rat at different times during the post-mortem period, and theoretically justified the observed phenomena. The results obtained allow us to consider the studied indicators as a potential criterion for determining the post-mortem interval.

Clinical associations of host genetic variations in the genes of cytokines in critically ill patients.

Host genetic variations may influence a changing profile of biochemical markers and outcome in patients with trauma/injury. The objective of this study was to assess clinical associations of single nucleotide polymorphisms (SNPs) in the genes of cytokines in critically ill patients. A total of 430 patients were genotyped for SNPs in the genes of pro- (IL1B, IL6, IL8) and anti-inflammatory (IL4, IL10, IL13) cytokines. The main end-points were sepsis, mortality and adult respiratory distress syndrome (ARDS). We evaluated the dynamic levels of bilirubin, blood urea nitrogen, creatine kinase, creatinine and lactate dehydrogenase in five points of measurements (between 1 and 14 days after admission) and correlated them with SNPs. High-producing alleles of proinflammatory cytokines protected patients against sepsis (IL1B -511A and IL8 -251A) and mortality (IL1B -511A). High-producing alleles of anti-inflammatory cytokines IL4 -589T and IL13 431A (144Gln) were less frequent in ARDS patients. The carriers of IL6 -174C/C genotypes were prone to the increased levels of biochemical markers and acute kidney and liver insufficiency. Genotype-dependent differences in the levels of biochemical indicators gradually increased to a maximal value on the 14th day after admission. These findings suggest that genetic variability in pro- and anti-inflammatory cytokines may contribute to different clinical phenotypes in patients at high risk of critical illness.

[INHALED ANTIBIOTICS IN TREATMENT OF NOSOCOMIAL PNEUMONIA].

Nosocomial pneumonia is the most common infection in intensive care units. Currently the problem of resistance of noso-comial pathogens to miost of antibiotics is crucial. Using of inhaled antibiotics in combination with intravenous drugs is eff ective and safe method for treatment of nosocomial pneumonia. The literature review describes current opportunities of ihhaled antibiotic therapy of nosocomial pneumonia, descriptions of drugs, the advantages and disadvantages of this treatment. Special attention is paid for using inhaled aminoglycosides for nosocomial pneumonia.

[Pathogenesis and target therapy of acute respiratory distress syndrome].

The paper summarizes results of experimental studies and clinical observations of the pathogenesis and effectiveness of respiratory, non-respiratory and pharmacological treatment methods for acute respiratory distress syndrome caused by direct and indirect damaging factors. The article deals with differences and peculiarities of morphological changes and lung functional disorders, clinical, laboratory and instrumental signs of various origins in ARDS and justifies necessity of differential diagnosis and differential treatment of ARDS, depending on the reasons for its development. Furthermore the article discusses an algorithm for differential diagnosis and differential treatment for ARDS caused by direct and indirect damaging factors.

[Genetic study of predisposition to community-acquired pneumonia].

The study included 243 patients with acute community-acquired pneumonia and 173 healthy subjects. The following candidate loci were used to investigate genetic variability: 3 sites of CYP1A1, GSTM1, GSTT1, GSTP1, ACE gene of the rennin-angiotensin system, chemokine receptor gene CCR5. Enhanced predisposition to pneumonia was shown to be characteristic of homozygotes in deletion at the ACE locus (OR = 1.8; p = 0.013), carriers of normal alleles of the GSTM1 locus (OR = 1.7; p = 0.010), and homozygotes in allele 606T of the CYP1A1 gene (OR = 1.6; p = 0.020).

[Lung morphological changes in premature neonates with hyaline membrane disease due to the use of exogenous surfactants during mechanical ventilation].

A complex of studies was used in 4 groups of premature babies to study lung tissue morphological changes in hyaline membrane disease, by applying exogenous surfactants during mechanical ventilation. Background diseases, pre- and intranatal risk factors, the babies' longevity, and the specific features of lung tissue and forming hyaline membranes were ascertained. Exogenous surfactants were found to have a blocking effect on the formation of hyaline membranes under mechanical ventilation.

[Intracardiac hemodynamic changes in the newborns with respiratory distress syndrome].

The paper provides the results of intracardiac circulation ultrasound study in 37 preterm neonatal infants, including 24 patients with severe respiratory distress syndrome (RDS), receiving the exogenous surfactant Curosurf in the complex therapy of the disease. A control comprised 12 apparently healthy preterm neonates who had no clinical signs of RDS in the early adaptive period or artificial ventilation (AV). Both groups were similar in the major anthropometric characteristics and gestational age. The objective of this investigation was to make Doppler echocardiographic study of blood flow through all cardiac valves in the newborn with RDS during AV. The investigation indicated that the neonates with severe RDS had increases in peak blood flow velocity and in peak pressure gradient through the valves of the great vessels: the aorta and pulmonary trunk, and abnormal regurgitation flow mainly through the pulmonary arterial valve, which was a sign of intensive hemodynamic adaptation in the acute phase of disease. By the third day of life, some neonatal infants without clinical signs of RDS were observed to have signs of intensive hemodynamic adaptation: increases in peak blood flow velocity and in peak pressure gradient through the valves of the pulmonary trunk. Irrespective of the specific features of the course of an early neonatal period, neonatal infants need Doppler echocardiographic monitoring for the evaluation of intracardiac hemodynamics.

[Morphology of acute lung injuries in mechanic trauma].

The lungs from 60 subjects who had died of polytrauma were studied morphologically. The heads of the corpses were not injuried. The aim of the study was investigation of characteristics and time of development of structural changes associated with lung injury. Early structural changes in trauma were disorders of circulation including microcirculation, acute emphysema, distelectases and atelectases, injury of bronchial and bronchiolar mucosa. Pulmonary edema and systemic inflammatory reaction emerge in the first hours after trauma.

Crystallization of alpha-galactosidase from Trichoderma reesei.

An extracellular alpha-galactosidase was isolated from fungus Trichoderma reesei. The purified enzyme had a molecular weight of 54,000 Da and an isoelectric point of 5.25. Crystals of alpha-galactosidase were obtained from a polyethylene glycol 4000 solution by the hanging-drop method. Seeding was used for enlargement of the crystal size. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 122.2 A, b = 81.0 A c = 49.7 A and diffract beyond 3.0 A resolution.

Crystals of beta-xylanase from Aspergillus oryzae.

An endo-xylanase was isolated from the culture of fungus Aspergillus oryzae variant D5. The purified enzyme had a molecular weight of 24,000 and the isoelectric point of 3.6. Xylanase crystals were obtained from a polyethylene glycol 6000 solution by the hanging-drop method. Seeding was used for the enlargement of the crystal size. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 54.9 A, b = 74.5 A, c = 50.8 A, and beta = 108.7 degrees. Crystals diffract beyond 2.5 A resolution.

Crystallization and preliminary X-ray study of minor glucoamylase from Aspergillus awamori variant X-100/D27.

Crystals of the reduced form of glucoamylase were obtained from polyethylene glycol 6000 solution by the hanging-drop method. The protein was treated with alpha-mannosidase to partly remove the sugar component. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 116.7 A, b = 104.3 A, c = 48.5 A and diffract beyond 2.5 A resolution.

Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

[Dynamic morphological changes observed in the respiratory system immediately after craniocerebral trauma].

Microscope and morphometry examinations of specific structural changes observed in the respiratory system in death immediately after severe craniocerebral trauma denoted profound lesions with a certain sequence of progression. The extent of histomorphological changes in the lungs is shown to be influenced by a life span of victims after inflicted trauma, i.e. by the prescription of trauma.

Effect of modification of carbohydrate component on properties of glucoamylase.

In this study, we investigated enzymatic deglycosylation of glucoamylase from Aspergillus awamori X 100/D27, a glycoprotein which has two N-linked and about forty short mannose-bearing O-linked sugars per molecule. O-Linked sugars were modified by treatment with alpha-mannosidase and N-linked sugars were removed using endo-beta-N-acetylglucosaminidase F. Analysis of conformational changes following deglycosylation suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase domains. Modification of the carbohydrate component by adding 1-deoxymannojirimycin to the culture medium induced inhibition of alpha-mannosidases involved in the processing, leading to a more complete glycosylation and, consequently, to a higher stability of the enzyme.

Transglycosylation activity of alpha-D-galactosidase from Trichoderma reesei. An investigation of the active site.

The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.

[The role of oligophosphate and nucleoside fragments upon the interaction of a nucleotide with RecA protein]. / Rol' oligofosfatnogo i nukleozidnogo fragmentov pri vzaimodeistvii nukleotida s belkom RecA.

Pyrophosphate and 1-pyrophospho-5-phosphoribose displace the nucleotide from the complex with RecA protein (in the absence of DNA). Adenosine, AMP, inorganic phosphate riboso-5-phosphate have no effect. Pyrophosphate causes the dissociation of RecA-ssDNA-complex in the same manner as ADP, circular hexaphosphate has no effect. In the complex with RecA protein the dissociation constant for pyrophosphate is 1.6 mM and 710 mM for adenosine. It is proposed that the oligophosphate is the main binding group in the RecA-nucleotide interaction. Two vicinal "closed" -O and one terminal "open" oxygenous =O-groups of oligophosphate take part in the binding. The nucleoside fragment alone cannot provide the firm binding of the nucleotide with the protein, but probably is responsible for the specificity of the nucleotide to protein binding.

[The biomicroscopic evaluation of the blood microcirculatory bed in the mesentery of the small intestine during experimental perftoran infusion under normovolemia]. / Biomikroskopicheskaia otsenka gemomikrotsirkuliatornogo rusla bryzheiki tonkoi kishki pri éksperimental'noi infuzii perftorana v usloviiakh normovolemii.

Dynamics of biomicroscopic state of hemomicrocirculatory bed (HMCB) of small intestinal mesentery was studied in go both sexes albino outbred rats in conditions of intravenous administration of 1.0 ml perfuoram (PF) with simultaneous control of the experiment by intravenous isotonic solution administration. Biomicroscopy was performed 30 minutes, 3, 6 and 24 hours later. 30 minutes after the PF administration bloodflow reduction in afferent vessels, elongation of construction period of resistant vessels and 18% decrease of vessels were demonstrated. Precapillary diameter decreased on 23% with respect to control with isotonic solution. Tortuosity of precapillaries and decrease of functioning capillaries number. 3 hours after the PF infusion blood flow turned more slow, signs of adhesion between leukocytes and postcapillary venules lumenal surface appeared and perivascular infiltrate had formed. Diameter of alducent vessels reduced in comparison with control and went lugher against the 30-minutes research stage. On the whole bloodflow in resistant vessels turns homogeneous. 6 hours after PF infusion homogeneous bloodflow could be seen. Number of arteriolar vasomotions grew up to control and made 14 in a minute. Venular diameter was close to control. Biomicroscopy of HMCB 24 hours after infusion did not reveal essential differences compared to with isotonic solution administered.