ABSTRACT
The aim of the study was to assess the functional activity of peripheral blood phagocytes in patients with drug-induced osteonecrosis of the jaws to develop a new approach to treatment. A total of 35 people were examined, including: 10 healthy individuals, 8 with purulent-inflammatory diseases of the maxillofacial region (GVZ ChLO), 17 patients with medical osteonecrosis of the jaws (MRONJ). Among patients with MRONJ, there were 10 men and 7 women, aged from 54 to 73 years, with a mean age of 62 years. She had a history of cancer (prostate cancer, breast cancer). In connection with the presence of metastases, the patients took bisphosphonates - the drug Zometa (zoledronic acid) in injections of 4 mg intravenously 1 time per month. Phagocytic activity of peripheral blood leukocytes was determined by flow cytometry. The examination of the patients was carried out before and on the 14th day after the surgical treatment, osteonecrectomy. In patients with MRONJ and HVD, the phagocytic activity of leukocytes was reduced by 30-40% compared with healthy individuals (p<0.05). Low phagocytic activity was confirmed by the indicators of stimulation of E. coli leukocytes and the stimulation index (oxidative burst). On the 14th day after surgical treatment in patients with BRONJ, the leukocyte phagocytosis indices did not normalize. The dysfunction of macrophage cells is more pronounced in cancer patients treated with bisphosphonates than in patients with HVD ChLH. A low level of indices of innate immunity factors contributes to the recurrence of osteonecrosis.
Subject(s)
Bisphosphonate-Associated Osteonecrosis of the Jaw , Bone Density Conservation Agents , Bone Density Conservation Agents/adverse effects , Escherichia coli , Female , Humans , Jaw , Leukocytes , Male , Middle Aged , Neoplasm Recurrence, LocalABSTRACT
The aim of the study was to develop a new approach to the treatment of patients with drug-induced osteonecrosis of the jaws based on the correction of disorders of the oral microbiota. We examined 22 patients with MRONJ and 10 healthy individuals aged 55 to 76 years. All patients underwent surgical treatment for cancer, in connection with tumor metastasis, they took bisphosphonates - the drug Zometa (zoledronic acid) in injections of 4 mg intravenously 1 time per month. To assess the microbiota of the oral cavity, we used the method of chromatography-mass spectrometry (CMS) of microbial markers before and after osteonecrectomy. Patients with MRONJ in the oral cavity showed high rates of aerobic-anaerobic associations, actinobacteria, Candida fungi and viruses, the number of which was 2-80 times higher than in healthy individuals. After surgical treatment, the indices returned to normal only in mild MRONJ patients. The level of normal flora in the oral microbiota in patients with MRONJ was characterized by low levels of Bifidobacterium spp. After 6 months. after surgical treatment, relapses occur in 25% of patients with mild course and in 71% of patients with severe course of MRONJ. The new algorithm of complex treatment consists in carrying out drug treatment in the postoperative period, including long-term use of local antiseptics with a pronounced complex antibacterial, antifungal and antiviral effect, and probiotics containing bifidobacteria.
Subject(s)
Bisphosphonate-Associated Osteonecrosis of the Jaw , Bone Density Conservation Agents , Algorithms , Bisphosphonate-Associated Osteonecrosis of the Jaw/drug therapy , Bisphosphonate-Associated Osteonecrosis of the Jaw/surgery , Diphosphonates , Humans , JawABSTRACT
The goal of the study is assessing the population structure of lymphocytes and the subpopulation composition of NK cells in the peripheral blood of humans in the case of drug-induced osteonecrosis of the jaw for improving the quality of diagnosis, treatment, and prevention of osteonecrosis of the jaw. Thirty patients were examined, including 15 patients with drug-induced osteonecrosis of the jaw (ONJ), 10 healthy individuals, and five patients with pyoinflammatory diseases of the maxillofacial region, aged 49 to 77. Every four weeks, the patients had been receiving 4 mg intravenous injections of bisphosphonates (the Zometa preparation (zoledronic acid)) for 1.5 - 3 years. The study was performed on a FACS Canto II flow cytometer manufactured by Becton Dickinson (BD), USA. In the patients with ONJ, an imbalance of the NK cell subtypes was observed. As to the common NK cells, the level of subtypes of cytolytic NK cells (CD3-CD16+(or hight)56dim) was elevated, and the level of cytokine-producing cells (СD3-CD16-(or low) 56bright) was reduced, compared to the healthy individuals (p<0.05). In the patients with ONJ, after the surgical treatment, the relative and absolute levels of lymphocytes and the total level of NK cells (CD3-CD16+/56+) normalized on the seventh day. The level of NK cells subtypes did not change after the treatment. This pathology is characterized by a low level of innate protection factors, as evidenced by the reduction of the total population of NK cells, and the imbalance of NK cells subtypes. The imbalance of NK cells (the natural killer cells) was an indicator of unfavorable prognosis for osteonecrosis treatment.
Subject(s)
Bisphosphonate-Associated Osteonecrosis of the Jaw , Bone Density Conservation Agents , Jaw Diseases , Killer Cells, Natural , Lymphocytes , Aged , Bisphosphonate-Associated Osteonecrosis of the Jaw/immunology , Diphosphonates/adverse effects , Humans , Middle AgedABSTRACT
In patients with drag-induced jaw osteonecrosis which developed in cancer patients due to administration of bisphosphonates for metastases, the microflora of the oral cavity was examined by chromatography-mass spectrometry (HMS). The method of HMS can be used as an additional method of diagnostics in complex clinical cases with ineffective treatment.
Subject(s)
Bone Density Conservation Agents , Jaw Diseases , Osteonecrosis , Bone Density Conservation Agents/adverse effects , Diphosphonates , Humans , Jaw , Jaw Diseases/chemically induced , Jaw Diseases/diagnostic imaging , Mass Spectrometry , Osteonecrosis/chemically induced , Osteonecrosis/diagnostic imagingABSTRACT
Substitution of mandibular defects resulting from injuries and inflammatory bone diseases as well as surgical interventions for tumors is an important medical and scientific problem. The article presents the results of surgical treatment of 8 patients with the use of combined endoprosthesis of the mandible consisting of individual titanium metal structure and titanium silk. This solution provides the possibility for reliable fixation of the soft tissue structures of the lower face region by suturing, including facial and masticatory muscles, improving the biological integration of the endoprosthesis. The combined design shows high efficiency of restoration of both aesthetic and functional characteristics of the mandible, as well as good long term survival rate.
Subject(s)
Dental Implants , Mandibular Reconstruction , Humans , Mandible , Masticatory Muscles , TitaniumABSTRACT
The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35â Å resolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.
Subject(s)
Laccase/metabolism , Oxygen/metabolism , Polyporales/enzymology , Water/metabolism , Biocatalysis/radiation effects , Catalytic Domain/radiation effects , Crystallography, X-Ray , Laccase/chemistry , Models, Molecular , Oxidation-Reduction/radiation effects , Polyporales/chemistry , Polyporales/radiation effects , Protein Conformation/radiation effects , X-RaysABSTRACT
Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68â Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.
Subject(s)
Bacterial Proteins/chemistry , Shewanella/enzymology , Uridine Phosphorylase/chemistry , Catalytic Domain , Crystallography, X-Ray , Hydrogen Bonding , Kinetics , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Substrate Specificity , Uridine/chemistryABSTRACT
Laccases belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water without the formation of hydrogen peroxide. The activity of laccases depends on the number of Cu atoms per enzyme molecule. The structure of type 2 copper-depleted laccase from Botrytis aclada has been solved previously. With the aim of obtaining the structure of the native form of the enzyme, crystals of the depleted laccase were soaked in Cu(+)- and Cu(2+)-containing solutions. Copper ions were found to be incorporated into the active site only when Cu(+) was used. A comparative analysis of the native and depleted forms of the enzymes was performed.
Subject(s)
Botrytis/enzymology , Copper/metabolism , Laccase/metabolism , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Ions , Laccase/chemistry , Models, Molecular , Molecular Sequence Data , TemperatureABSTRACT
Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.
Subject(s)
Cytochromes a1/chemistry , Cytochromes a1/metabolism , Cytochromes c1/chemistry , Cytochromes c1/metabolism , Ectothiorhodospiraceae/enzymology , Heme/chemistry , Nitrate Reductases/chemistry , Nitrate Reductases/metabolism , Nitrites/metabolism , Protein Conformation/radiation effects , Sulfites/metabolism , Catalytic Domain , Crystallography, X-Ray , Cytochromes a1/radiation effects , Cytochromes c1/radiation effects , Ectothiorhodospiraceae/radiation effects , Models, Molecular , Nitrate Reductases/radiation effects , Nitrites/chemistry , Nitrites/radiation effects , Protein Binding , Radiation Effects , Substrate Specificity , Sulfites/chemistry , Sulfites/radiation effects , X-RaysABSTRACT
Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.
Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Poly I/metabolism , Bacillus/chemistry , Bacillus/enzymology , Bacillus/genetics , Catalysis , Cell Line, Tumor , Cell Survival/drug effects , Endoribonucleases/metabolism , Humans , Kinetics , Models, Molecular , Mutagenesis , Mutant Proteins/metabolism , X-Ray DiffractionABSTRACT
The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.
Subject(s)
Endo-1,4-beta Xylanases/genetics , Endo-1,4-beta Xylanases/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Penicillium/enzymology , Penicillium/genetics , Recombinant Proteins/metabolism , Symporters/chemistry , Symporters/metabolism , Crystallography, X-Ray , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/isolation & purification , Escherichia coli Proteins/genetics , Recombinant Proteins/genetics , Substrate Specificity , Symporters/geneticsABSTRACT
Prolidases are peptidases that are specific for dipeptides with proline as the second residue. The structure of recombinant prolidase from the hyperthermophilic archaeon Thermococcus sibiricus (Tsprol) was determined at 2.6â Å resolution. The homodimer of Tsprol is characterized by a complete lack of interactions between the N- and C-terminal domains of the two subunits and hence can be considered to be the most open structure when compared with previously structurally studied prolidases. This structure exists owing to intermolecular coordination bonds between cadmium ions derived from the crystallization solution and histidine residues of a His tag and aspartate and glutamate residues, which link the dimers to each other. This linking leads to the formation of a crystal with a loose packing of protein molecules and low resistance to mechanical influence and temperature increase.
Subject(s)
Archaeal Proteins/chemistry , Dipeptidases/chemistry , Thermococcus/enzymology , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits/chemistry , Recombinant Proteins/chemistryABSTRACT
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02â Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.
Subject(s)
Adenosine Triphosphate/metabolism , DNA Ligases/chemistry , DNA, Archaeal/metabolism , Thermococcus/enzymology , Adenosine Triphosphate/chemistry , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Binding Sites , Crystallography, X-Ray , DNA Breaks, Single-Stranded , DNA Ligase ATP , DNA Ligases/metabolism , Models, Molecular , Protein Folding , Scattering, Small Angle , Thermococcus/classificationABSTRACT
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.
Subject(s)
DNA Ligases/chemistry , Thermococcus/enzymology , Crystallization , Crystallography, X-Ray , DNA Ligase ATP , DNA Ligases/genetics , DNA Ligases/isolation & purification , Enzyme Stability , Gene Expression , TemperatureABSTRACT
Alcohol dehydrogenases belong to the oxidoreductase family and play an important role in a broad range of physiological processes. They catalyze the cofactor-dependent reversible oxidation of alcohols to the corresponding aldehydes or ketones. The NADP-dependent short-chain alcohol dehydrogenase TsAdh319 from the thermophilic archaeon Thermococcus sibiricus was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method using 25%(w/v) polyethylene glycol 3350 pH 7.5 as precipitant. The crystals diffracted to 1.68 A resolution and belonged to space group I222, with unit-cell parameters a = 55.63, b = 83.25, c = 120.75 A.
Subject(s)
Alcohol Dehydrogenase/chemistry , Thermococcus/enzymology , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/isolation & purification , Crystallization , Crystallography, X-Ray , Enzyme Stability , Gene Expression , TemperatureABSTRACT
NAD(+)-dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD(+) to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95 A resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD(+)-dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374-399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation.
Subject(s)
Formate Dehydrogenases/chemistry , Moraxella/enzymology , Amino Acid Sequence , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Biocatalysis , Crystallography, X-Ray , Formate Dehydrogenases/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Substrate SpecificityABSTRACT
It is supposed that alpha,gamma-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA polymerase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K(m) values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K(m)(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg2+ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.
Subject(s)
Aminobutyrates/chemistry , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Aminobutyrates/chemical synthesis , Catalytic Domain , Enzyme Inhibitors/chemical synthesis , Kinetics , Phenylbutyrates , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
The crystal structure of the ternary complex of NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. C-1 with the cofactor (NAD+) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacitalicents parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the titalicperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreitalicent with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction.