Crystal-packing analysis of translation initiation factor 2 reveals new details of its function.

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

Photodynamic Inactivation of Mycobacterium tuberculosis Using Alluminium Phthalocyanine.

In a series of in vitro experiments, the optimum regimes of laser treatment were determined for effective photodynamic inactivation of Mycobacterium tuberculosis at a constant dose of aluminum phthalocyanine. Reference laboratory drug-susceptible strain H37Rv and clinical isolates of M. tuberculosis with varying degrees of resistance to antibiotics were used. Suspensions of M. tuberculosis were incubated with aluminum phthalocyanine in a concentration of 5 µg/ml and then subjected to photodynamic inactivation with high- or low- intensity laser irradiation at λ=662 nm at various parameters of light power density. Mycobacteria survival rate was assessed by CFU assay on solid media. It was shown that at the specified dose of the photosensitizer, the photodynamic inactivation of mycobacterium was characterized by inhibition and complete cessation of their growth depending on the dose density of the laser energy. Effective photodynamic inactivation started from a light dose density of 46.9 J/cm2 at a radiation power of 0.01 W and from 56.25 J/cm2 at a radiation power of 0.1 W. Photodynamic inactivation at low laser power is more effective against drug-susceptible strains of M. tuberculosis.

[Influence of Nonconserved Regions of L1 Protuberance of Thermus thermophilus Ribosome on the Affinity of L1 Protein to 23s rRNA].

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.

Perfect Hemihedral Twinning in Crystals of the γ-Subunit of Translation Initiation Factor 2 from Sulfolobus solfataricus: Cause and Effect.

The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus (SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration. Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γ- and α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

High-resolution crystal structure of the isolated ribosomal L1 stalk.

The crystal structure of the isolated full-length ribosomal L1 stalk, consisting of Thermus thermophilus ribosomal protein L1 in complex with a specific 80-nucleotide fragment of 23S rRNA, has been solved for the first time at high resolution. The structure revealed details of protein-RNA interactions in the L1 stalk. Analysis of the crystal packing enabled the identification of sticky sites on the protein and the 23S rRNA which may be important for ribosome assembly and function. The structure was used to model different conformational states of the ribosome. This approach provides an insight into the roles of domain II of L1 and helix 78 of rRNA in ribosome function.

Structural analysis of interdomain mobility in ribosomal L1 proteins.

Ribosomal protein L1 consists of two domains connected by two oppositely directed fragments of the polypeptide chain in a hinge-resembling fashion. The domain arrangement determines the overall shape of the protein, corresponding to an open or a closed conformation. Ribosomal L1 proteins from archaea demonstrate the open conformation in both isolated and RNA-bound forms. RNA-free ribosomal L1 proteins from bacteria display the closed conformation, whereas in complex with RNA these proteins exist in an open conformation similar to their archaeal counterparts. Analysis of all available L1 amino-acid sequences shows that in comparison to the archaeal proteins, the bacterial proteins possess an extra residue in one of the two interdomain fragments which could be responsible for their closed conformation. To verify this suggestion, a Thermus thermophilus L1 mutant lacking one residue in the fragment corresponding to the hinge was obtained and its crystal structure was solved. It was found that this mutation transformed the closed conformation of the bacterial L1 protein into an open conformation similar to that of the archaeal L1 proteins.

[Compression syndromes in the disc-radicular conflict in patients with intervertebral disc lesion].

One thousand and thirty patients with proved L(5)/S(1)disc herniations were examined. The dynamics of disease in acute stage was studied using ultrasonography and electromyography in 3 groups of patients: 280 with lumboischialgia, 520 with the root syndrome and 230 controls without back pain. It has been shown that edematous epiduritis and venous stas in the superincumbent spinal movement segment is a main pathogenetic mechanism of acute phase of the disc-radicular conflict. Focal myelopathy is a pathogenetic mechanism of radiculopathy in the disc-recticular conflict. Compression and reflex syndromes are caused by the common mechanisms and their differentiation by clinical presentations has a conditional character.

[Efficiency of lymphotropic intermittent chemotherapy in the complex treatment of patients with progressive pulmonary tuberculosis].

The study has established that during a progressive tuberculous process, immunological changes appear as significant increased proinflammatory cytokine production that leads to progressive destructive processes in the lung parenchymal lesion areas. Inclusion of lymphotrophic chemoimmunotherapy with roncoleukin into the complex therapy of patients with progressive pulmonary tuberculosis permits the optimization of the results of treatment in patients with progressive pulmonary tuberculosis due to the target delivery of antituberculous and immunoactive agents.

Three-dimensional structures of L-asparaginase from Erwinia carotovora complexed with aspartate and glutamate.

The crystal structures of Erwinia carotovora L-asparaginase complexed with L-aspartate and L-glutamate were determined at 1.9 and 2.2 A, respectively, using the molecular-replacement method and were refined to R factors of about 21% in both cases. The positions of the ligands in the active site were located. A comparison of the new structures with the known structures of Escherichia coli L-asparaginase and Er. chrysanthemi L-asparaginase was performed. It was found that the arrangement of the ligands practically coincides in all three enzymes. The peculiarities of the quaternary structure of the enzyme, the possible role of water molecules in the enzyme action and the conformational changes during the catalyzed reaction are discussed.

[Crystal structures of mutant ribosomal proteins L1].

Nine mutant forms of ribosomal proteins L1 from the bacterium Thermus thermophilus and the archaeon Methanococcus jannaschii were obtained. Their crystal structures were determined and analyzed. Earlier determined structure of S179C TthL1 was also thoroughly analyzed. Five from ten mutant proteins reveal essential changes of spatial structure caused by surface point mutation. It proves that for correct studies of biological processes by site-directed mutagenesis it is necessary to determine or at least to model spatial structures of mutant proteins. Detailed comparison of mutant L1 structures with that of corresponding wild type proteins reveals that side chain of a mutated amino acid residue tries to locate like the side chain of the original residue in the wild type protein. This observation helps to model the mutant structures.

[Characteristics of A-interferon-generated dendritic cells in patients with pulmonary tuberculosis].

The phenotype of the dendritic cells (DC) generated from the adhesion fraction of mononuclear cells in the presence of GM-CSF and alpha-interferon was studied in patients with pulmonary tuberculosis. Despite the absence of significant differences in the count of mature CD83+DCs in the groups of patients (n = 38) and healthy donors (n = 30), elevated CD14(+)-monocyte levels and few activated CD25(+)-DCs were indicative of the impaired process of DC maturation/generation in patients with pulmonary tuberculosis, particularly in a subgroup of patients with a low T-cell proliferative response against PPD (PPD-anergy, n = 10). The patients with tuberculosis showed the lower relative levels of CD11c(-)-CD123(+)-DC and the normal levels of myeloid CD11c(+)D123(-)DCs. However, in patients with PPD-anergy, the content of myeloid CD11c(+)CD123(-)-DCs was significantly higher than that in PPD-reactive patients. Moreover, the patients with PPD-anergy were characterized by the elevated peripheral blood levels of CD14+CD16(+)-monocytes, which was associated with the high suppressive activity of monocytes (r(s) = 0.53; p < 0.05). The impaired process of DC generation/maturation in patients with pulmonary tuberculosis is believed to be associated with the changes in the phenotypic and functional properties of monocytes and to be a cause of an inadequate antigen-specific response in tuberculous infection.

Autologous bone marrow cells in the treatment of cirrhosis of the liver.

The data characterizing tolerance and efficiency of autologous bone marrow cells in the treatment of patients with cirrhosis of the liver are presented. Injection of autologous bone marrow cells was not associated with the development of adverse reactions. Cell therapy of patients with compensated cirrhosis arrested asthenic syndrome, reduced cytolysis, increased the level of serum albumin and platelet count. Ultrasonic examination revealed reduction of portal hypertension (the area of the spleen and the portal vein lumen decreased). In patients with decompensated cirrhosis, a positive response presenting as reduction of the disease severity (by 1.9 points) was observed in 48.6% cases. Positive shifts in these patients were associated with a decrease of ALT and AST levels, reduction of laboratory signs of cirrhosis, increase in platelet count, and reduction of the asthenic syndrome. Hence, therapy with autologous bone marrow cells is safe and, according to preliminary results, can be regarded as a new approach to the treatment of patients with cirrhosis of the liver.

Use of interferon-alpha-induced dendritic cells in the therapy of patients with malignant brain gliomas.

Clinical and immunological analysis of the efficiency of combined immunotherapy with the use dendritic cells for the treatment of malignant glioma of the brain was carried out. Dendritic cells generated in the presence of granulocyte-macrophage CSF and IFN-alpha retain their functional characteristics in patients with gliomas, which suggests the possibility of their use for the treatment of malignant tumors (glioma) of the brain. Combined therapy using interferon-induced dendritic cells was associated with generation of antigen-specific immune response during vaccinations. The results indicate satisfactory tolerance of combined immunotherapy using dendritic cells and the absence of toxic side effects at the stage of adoptive immunotherapy and at the stage of vaccinations with dendritic cells. Clinical trials showed that vaccinations with dendritic cells included into combined immunotherapy improved the quality of life and survival of patients with malignant gliomas.

Structure of the ribosomal protein L1-mRNA complex at 2.1 A resolution: common features of crystal packing of L1-RNA complexes.

The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1 A resolution. It was found that all polar atoms involved in conserved protein-RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K(+) ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1-RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open-open or open-closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation.

[Interactions of ribosomal protein L1 with ribosomal and messenger RNAs].

Crystal structures of unbound protein L1 and of its complexes with ribosomal an messenger RNAs are analyzed. It is shown that the values of the apparent association rate constant for L1-RNA depend on conformation of unbound protein L1. It is suggested that L1 binds to rRNA with higher affinity than to mRNA because of additional interactions between domain II of L1 and the loop rRNA region, which is absent in mRNA.

[Interleukin-2 in the correction of T-cell anergy in patients with pulmonary tuberculosis].

The clinical and immunomodulating effects of lymphotropic administration of interleukin-2 (IL-2) were studied in the combine treatment of patients with pulmonary tuberculosis. The patients with tuberculosis were shown to have the low levels of monocytes with the intracellular expression of tumor necrosis factor-alpha (TNF-alpha) and the high count of CD14+ CD16+ monocytes with the intracellular expression of IL-10. The changes in the monocytic link were most pronounced in patients with PPD-induced anergy appeared as the low proliferation and production of alpha-interferon (alpha-INF). During clinical trials, 19 patients received tuberculostatic therapy in combination with IL-2 (Roncoleukin) (a study group) whereas 16 patients had tuberculostatic therapy alone (a control group). The administration of Roncoleukin statistically significant increased a proliferative response to PPD and normalized the count of CD14+ CD16+ monocytes with anti-inflammatory and immunosuppressive activities. The restoration of a PPD response was recorded more frequently in the study group than in the control one (75% vs 30%; p = 0.045). The magnitude of positive X-ray changes was also higher in the study group than in the control one (63% vs 25%; p = 0.026). The findings suggest the clinical and immunomodulating effects of IL-2 (Roncoleukin) in the combined therapy for tuberculosis.

Structure of Escherichia coli glutamate decarboxylase (GADalpha) in complex with glutarate at 2.05 angstroms resolution.

Glutamate decarboxylase (GAD) is a pyridoxal enzyme that catalyzes the conversion of L-glutamate into gamma-aminobutyric acid and carbon dioxide. The Escherichia coli enzyme exists as two isozymes, referred to as GADalpha and GADbeta. Crystals of the complex of the recombinant isozyme GADalpha with glutarate as a substrate analogue were grown in space group R3, with unit-cell parameters a = b = 117.1, c = 196.4 angstroms. The structure of the enzyme was solved by the molecular-replacement method and refined at 2.05 angstroms resolution to an R factor of 15.1% (R(free) = 19.9%). The asymmetric unit contains a dimer consisting of two subunits of the enzyme related by a noncrystallographic twofold axis which is perpendicular to and intersects a crystallographic threefold axis. The dimers are related by a crystallographic threefold axis to form a hexamer. The active site of each subunit is formed by residues of the large domains of both subunits of the dimer. The coenzyme pyridoxal phosphate (PLP) forms an aldimine bond with Lys276. The glutarate molecule bound in the active site of the enzyme adopts two conformations with equal occupancies. One of the two carboxy groups of the glutarate occupies the same position in both conformations and forms hydrogen bonds with the N atom of the main chain of Phe63 and the side chain of Thr62 of one subunit and the side chains of Asp86 and Asn83 of the adjacent subunit of the dimer. Apparently, it is in this position that the distal carboxy group of the substrate would be bound by the enzyme, thus providing recognition of glutamic acid by the enzyme.

Structure of Citrobacter freundii L-methionine gamma-lyase.

L-Methionine gamma-lyase (MGL) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes gamma-elimination of L-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.

[Identification of RNA-recognizing modules on the surface of ribosomal proteins]. / Identifikatsiia RNK-uznaiushchikh modulei na poverkhnosti ribosomnykh belkov.

Properties of specific interaction between ribosomal proteins and ribosomal RNAs were analyzed and a method for determination of "recognizing modules" on the protein surface was proposed. The method is based on the search of protein atoms making conserved H-bonds with RNA and forming an invariant spatial structure in homologous rRNA-protein complexes and in the isolated protein. A potential of the method is demonstrated on the determination of the recognizing modules on the surfaces of ribosomal proteins S8, S15 and L5.