Shock-Associated Systemic Inflammation in Amniotic Fluid Embolism, Complicated by Clinical Death.

Background: Amniotic fluid embolism (AFE) is one of the main causes of maternal mortality in developed countries. The most critical AFE variants may be considered from the perspective of systemic inflammation (SI), a general pathological process that includes high levels of systemic inflammatory response, neuroendocrine system distress, microthrombosis, and multiple organ dysfunction syndrome (MODS). This research work aimed to characterize the dynamics of super-acute SI using four clinical case studies of patients with critical AFE.

[Intersubunit Mobility of the Ribosome].

Ribosomes are ribonucleoprotein nanoparticles synthesizing all proteins in living cells. The function of the ribosome is to translate the genetic information encoded in a nucleotide sequence of mRNA into the amino acid sequence of a protein. Each translation step (occurring after the codon-dependent binding of the aminoacyl-tRNA with the ribosome and mRNA) includes (i) the transpeptidation reaction and (ii) the translocation that unidirectionally drives the mRNA chain and mRNA-bound tRNA molecules through the ribosomal intersubunit space; the latter process is driven by the free energy of the chemical reaction of transpeptidation.

Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the "initiation potential" of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture).

Formation of New Polysomes on Free mRNAs in a Cell-Free Translation Systems Is Accompanied by Partial Disassembly of Previously Formed Polysomes.

A method for detection of the fluorescence-labeled mRNA in translating ribosomal complexes has been developed. It is demonstrated that in the working cell-free translation system with preformed polysomes, formation of new polysomes on free mRNA takes place. For the first time, it is shown that the process is accompanied by partial disassembly of the previously formed polysomes.

Conformation transitions of eukaryotic polyribosomes during multi-round translation.

Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase').

The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes.

During protein synthesis, several ribosomes bind to a single messenger RNA (mRNA) forming large macromolecular assemblies called polyribosomes. Here we report the detailed molecular structure of a 100 MDa eukaryotic poly-ribosome complex derived from cryo electron tomography, sub-tomogram averaging and pseudo-atomic modelling by crystal structure fitting. The structure allowed the visualization of the three functional parts of the polysome assembly, the central core region that forms a rather compact left-handed supra-molecular helix, and the more open regions that harbour the initiation and termination sites at either ends.

Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study.

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein.

Internal translation initiation and eIF4F/ATP-independent scanning of mRNA by eukaryotic ribosomal particles.

The recombinant mRNAs with 5'-untranslated region, called omega leader, of tobacco mosaic virus RNA are known to be well translated in eukaryotic cell-free systems, even if deprived of cap structure. Using the method of primer extension inhibition (toe-printing), the ribosomal particles were shown to initiate translation at uncapped omega leader when its 5'-end was blocked by a stable RNA-DNA double helix, thus providing evidence for internal initiation. The scanning of the leader sequence and the formation of ribosomal 48S initiation complexes at the initiation AUG codon occurred in the absence of ATP-dependent initiation factor eIF4F, as well as without ATP.

Internal initiation of polyuridylic acid translation in bacterial cell-free system.

The task of the present work was to answer the question: is the free 5'-end needed for effective translation of a model polyribonucleotide template - polyuridylic acid - in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5'-end and the polyuridylic acid with blocked 5'-end were compared in the bacterial cell-free translation system. To block the 5'-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5'-end and uridylic oligoribonucleotide sequence at its 3'-end, schematically described as FAM(dC)10(rU)50, was covalently attached (ligated) to the 5'-end of the template polyuridylic acid.

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.

[Characteristics of septic shock in obstetrics].

Peculiarities of septic shock in obstetrics are considered in this article. Traits of pregnant woman organism concerning infectious process, etiology and risk factors, modern criteria of heavy sepsis diagnosis and septic shock are discussed. It is pointed out that septic process system manifestations considerably outstrips local manifestations of purulent process in a uterus and it is necessary to take into account modern markers and criteria when making decision on initial infection center sanitization.

[Monitoring of functional body status in agricultural machine operators].

Sanitary and hygienic work conditions of operators of different kinds of combine harvesters were evaluated. Pecularities of adaptation of labourers' organism to work process were studied. A technique of remote transmission of physiological data, obtained in the field conditions to remote laboratory for subsequent expert analysis of the results was used.

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.

Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation.

According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5'-end of mRNA and subsequently migrates along 5'-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5'-UTRs of different lengths and structures in mammalian and plant cell-free systems.

Insight into the structural organization of the omega leader of TMV RNA: the role of various regions of the sequence in the formation of a compact structure of the omega RNA.

The 5'-untranslated sequence of tobacco mosaic virus RNA--the so called omega leader--is a well-known translational enhancer. The structure of the omega RNA has unusual features. Despite the absence of extensive secondary structure of the Watson-Crick type, the omega RNA possesses a stable compact conformation.

Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA.

The 5'-untranslated sequence of tobacco mosaic virus RNA - the so-called omega leader - exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader.

Quantitative analysis of ribosome-mRNA complexes at different translation stages.

Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level.

Intramolecular triple helix as a model for regular polyribonucleotide (CAA)(n).

The regular (CAA)(n) polyribonucleotide, as well as the omega leader sequence containing (CAA)-rich core, have recently been shown to form cooperatively melted and compact structures. In this report, we propose a structural model for the (CAA)(n) sequence in which the polyribonucleotide chain is folded upon itself, so that it forms an intramolecular triple helix. The triple helix is stabilized by hydrogen bonding between bases thus forming coplanar triads, and by stacking interactions between the base triads.

[X-ray endovascular surgery in the trauma and orthopedic unit].

The review of X-ray endovascular surgical methods applied in the practice of FSF CITO named after N.N. Priorov since 1970 is presented.

Poly(A) leader of eukaryotic mRNA bypasses the dependence of translation on initiation factors.

Eukaryotic mRNAs in which a poly(A) sequence precedes the initiation codon are known to exhibit a significantly enhanced cap-independent translation, both in vivo and in cell-free translation systems. Consistent with high expression levels of poxviral mRNAs, they contain poly(A) sequences at their 5' ends, immediately before the initiation AUG codon. Here we show that poly(A) as a leader sequence in mRNA constructs promotes the recruitment of the 40S ribosomal subunits and the efficient formation of initiation complexes at cognate AUG initiation codons in the absence of two essential translation initiation factors, eIF3 and eIF4F.

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases.

Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation.

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation.

Continuous-exchange protein-synthesizing systems.

Protein synthesis in cell-free systems is an emerging technology already competing with in vivo expression methods. In this chapter the basic principles of continuous-exchange protein synthesizing systems, and protocols for Escherichia coli and wheat germ translation and transcription-translation systems are described. The ways to improve substrate supply in cell-free systems and mRNA design for eukaryotic system are discussed.