[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.

[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.

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.

[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.

Getting the most out of X-ray home sources.

The structures of a 14 kDa phospholipase, an 18 kDa proteinase inhibitor and a novel glycoside hydrolase with molecular weight 60 kDa were solved using the SAD technique and the effects of the amount of anomalous signal, completeness and redundancy of data on heavy-atom substructure determination, phasing and model building were analyzed. All diffraction data sets were collected on a Cu-anode X-ray home source. The structure of the phospholipase was obtained using the anomalous scattering contribution from its 16 S atoms. Three-dimensional models for the other two macromolecules were obtained using the anomalous contribution of I atoms rapidly incorporated into the crystal through the quick cryo-soaking method of derivatization. These results were used to discuss the application of sulfur- and iodine-SAD approaches in combination with X-ray home sources for high-throughput protein crystal structure solution. The estimates of the anomalous signal from S atoms in the gene products of four genomes are given and the prospects for increasing the anomalous contribution using longer wavelengths (e.g. from a chromium home source) and quick cryo-soaking derivatization are discussed. The possibility of rapidly preparing tangible home-source isomorphous derivatives suggests that this approach might become a valuable tool in the future of post-genomic projects.

[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.

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.

[Early morphological changes of the respiratory system after blunt multiple trauma]. / Morfologicheskie izmeneniia dykhatel'noi sistemy v rannem periode tupoi sochetannoi travmy.

Pronounced changes with a specific development sequence were detected on the basis of microscopy and morphometry examinations of peculiarities of structural changes in the respiratory system in death soon after mechanical trauma concomitant with head injuries. It was demonstrated the way the severity of histomorphologic changes occurring in the lungs influence the survival time of victims after trauma (time prescription).

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.

[Role of the structural changes of lungs in thanatogenesis of cranio-cerebral complicated trauma]. / Rol' strukturnykh izmenenii legkikh v tanatogeneze pri cherepno-mozgovoi i sochetannoi travme.

The lungs of 27 persons, who died of severe craniocerebral and combined trauma, were histologically examined. A deformation of the bronchi is registered in 15-60 min after craniocerebral trauma; desquamated epithelium and mucinous contents are found in their lumens. Erythrocytes and macrophages are detected in the alveolar lumens. The capillary alveolar septums are full-blooded; the alveolar septums are edematous with signs of hemorrhages and clusters of lymphoid cells. The venules are enlarged and full-blooded. Regions of the emphysema alternate with dystelectases and atelectases. The interstitial edema set on and the alveolar edema aggravates in 2-8 hours after trauma. There is an aggregation of erythrocytes. The leukocytic infiltration worsens. Hyaline membranes take shape. Similar changes are observed in the lungs in case of combined trauma; at the same time, the morphological changes are faster in the latter trauma variation. The detected structural impairments are typical of an acute pulmonary lesion and are crucial in thanatogenesis in craniocerebral and combined trauma.

[Effects of perftoran on experimental acute intestinal ischemia]. / Issledovanie éffektov perftorana na modeli ostroi intestinal'noi ishemii.

The studies were carried out on 76 albino noninbred male rats weighing 210-420 g. Ischemia of a jejunal segment was induced by applying a tourniquet to the base of a loop with the mesentery until the circulation fully stopped. Nonoxygenated perfluorane (an experimental group) or saline solution (a control group) was injected in a dose of 0.8-1.0 ml/100 g intraarterially 15 minutes before the termination of ischemia. During 90 min of reperfusion, there was a progressive decline of blood pressure (BP) with reference to the ischemic period by 39.8 +/- 20.9 and 20.8 +/- 14.6% in an experimental and control groups, respectively (p > 0.05). Biomicroscopy indicated that by min 5 of reperfusion, a relative reduction in the diameter (50-400 microns) of nutrient mesenteric arteries was half as large as that in the control group (24.0 +/- 5.5 and 45.1 +/- 3.6%; p < 0.05); by min 90, differences decreased (41.5 +/- 4.2 and 50.3 +/- 2.8%, respectively; p > or = 0.05). In the experimental group, perfluorane prevents the development of irreversible structural changes in the reperfusion period: the remaining cryptal epithelium is a source of regeneration of the integumentary epithelium of forming villi of the small intestinal mucosa.