[Histopathological evaluation of the evolution of oral mucosa grafts used for augmentation urethroplasty].

INTRODUCTION: The clinical efficiency of oral mucosa grafts used for augmentation urethroplasty has been proven. However, a small number of studies in literature are dedicated to the determination of pathologic changes in grafts during the engraftment in the urethra. AIM: to assess the histopathologic evolution of oral mucosa graft used for augmentation urethroplasty. MATERIAL AND METHODS: A total of 15 patients aged 19-67 years with penile and combined penile/bulbar urethral strictures of length 3.5-11 cm were undergone to two-staged urethroplasty by Asopa technique. In 9 cases the pathologic study of oral mucosa grafts after sampling (buccal mucosa in 5 cases and lip mucosa in 4 cases) and 6 months after their engraftment in the urethra during the second stage of urethroplasty was performed. In other 6 cases the graft tissue in patients with recurrent stricture that occurred 12-24 months after tubularization and requiring additional intervention was studied. RESULTS: The general structure of mucosal graft was preserved 6 months after sampling. The dystrophic changes in epithelium, inflammatory infiltration and foci of fibrosis in subepithelial layer were observed. Buccal grafts were distinguished by a smaller epithelium thickness and the presence of keratinization foci. The grafts also retained their structure after 12-24 months. A decrease in the severity of chronic inflammatory reaction and the absence of keratinization of the squamous buccal epithelium were found. The stricture in the area of anastomosis was characterized by the presence of sclerotic and fibrous connective tissue covered with urothelium. CONCLUSION: The oral mucosa grafts completely preserve their histologic structure during 1-2 years and they are not involved in recurrence of urethral stricture, which develops in the area of anastomosis between graft and native urethra.

Recombinant Antibodies to the Ebola Virus Glycoprotein.

Currently, there are no approved therapies for targeted prevention and treatment of Ebola hemorrhagic fever. In the present work, we describe the development of a eukaryotic expression system for the production of three full-length chimeric antibodies (IgG1-kappa isotypes) GPE118, GPE325, and GPE534 to the recombinant glycoprotein of the Ebola virus (EBOV GP), which is a key factor in the pathogenicity of the disease. The immunochemical properties of the obtained antibodies were studied by immunoblotting and indirect, direct, and competitive ELISA using the recombinant EBOV proteins rGPdTM, NP, and VP40. The authenticity of the antibodies and the absence of cross-specificity with respect to the structural proteins NP and VP40 of the Ebola virus were proved. The epitope specificity of the resulting recombinant antibodies was studied using commercial neutralizing antibodies against the viral glycoprotein. The recombinant antibodies GPE118, GPE325, and GPE534 were shown to recognize glycoprotein epitopes that coincide or overlap with the epitopes of three well-studied neutralizing anti-Ebola virus antibodies.

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL apical domain with implications for substrate interactions.

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (Tm>100 degrees C as evaluated by circular dichroism) is at least 35 degrees C greater than that of the E. coli apical domain (Tm=65 degrees C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 A using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 A based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the "Anfinsen cage" in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using 15N-1H correlation spectra of 15N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50 degrees C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal alpha-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.

Crystallization and preliminary X-ray investigation of holotryptophanases from Escherichia coli and Proteus vulgaris.

Crystals of Proteus vulgaris holotryptophanase have been grown by the hanging-drop technique using polyethylene glycol 4000 as precipitant in the presence of monovalent cations K+ or Cs+. Orthorhombic crystals (P2(1)2(1)2(1)) grown with Cs+ have unit cell parameters a = 115.0 A, b = 118.2 A and c = 153.7 A and diffract to 1.8 A. There are four subunits of the tetrameric molecule in the asymmetric unit. Native data have been collected to 2.5 A resolution. The 3.4 A data were collected from tetragonal crystals of Escherichia coli holotryptophanase grown under conditions described by Kawata et al. (1991). The molecular replacement solution for this crystal form has been found using tyrosine phenol-lyase coordinates. The correct enantiomorph is P4(3)2(1)2. There are two subunits in the asymmetric unit.

Crystal structure of tryptophanase.

The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.

The use of high doses of aprotinin to decrease blood loss in aortocoronary by-pass surgery.

There were examined 100 patients who had been operated in extracorporeal circulation for ischemic heart disease. In the 1st group (50 patients), each patient intraoperatively received 6,000,000 KIE of aprotinin; in the 2nd group (50 patients) placebo. Both groups were similar as for the age, duration of extracorporeal circulation, and myocardium anoxia. The total intraoperative blood loss was higher in 2nd group, 638 +/- 5 ml and 952.4 +/- 15.5 ml p < 0.05 correspondingly. The rate of cases without the intraoperative use of donor blood was 42% in group 1 and 18% in group 2. The postoperative blood loss was significantly lower with the use of aprotinin, 392 +/- 6.6 ml and 689.7 +/- 9.6 ml, p < 0.05 correspondingly; and in that the Hb concentration in the drainage blood was higher in group 2: 31.3 +/- 0.5 g and 73.1 +/- 2.2 g, p < 0.05. In group 1 22% of the patients received donor blood in the early postoperative period, and group 2 twice as many -56%. Thus prolonged intraoperative use of large aprotinin dosages in aortocoronary by-pass surgery is an effective means for stabilization of homeostasis, and it allows to reduce intra- and postoperative blood loss and diminishes the requirements in donor blood.

Recombinant Human Cyclophilin A in vitro Inhibits the Formation of Fibrin Clot.

The chemotactic properties of cyclophilin A are well-known. There exists however a poor level of understanding regarding the hemostatic effects of this protein. Herein it is shown that recombinant human cyclophilin A (rhСyA), in contrast to the granulocyte colony-stimulating factor, is capable of inhibitingin vitrothe formation of a fibrin clot, thereby violating the spatial dynamics of clot growth; this effect is transient and dose-independent. Furthermore, the hypothesis that the conformational changes in the thrombin-rhCyA complex may mediate the anticoagulant effect of rhCyA on the autowave processes of blood clotting is postulated.

Taking MAD to the extreme: ultrafast protein structure determination.

Multiwavelength anomalous diffraction data were measured in 23 min from a 16 kDa selenomethionyl substituted protein, producing experimental phases to 2.25 A resolution. The data were collected on a mosaic 3 x 3 charge-coupled device using undulator radiation from the Structural Biology Center 19ID beamline at the Argonne National Laboratory's Advanced Photon Source. The phases were independently obtained semiautomatically by two crystallographic program suites, CCP4 and CNS. The quality and speed of this data acquisition exemplify the opportunities at third-generation synchrotron sources for high-throughput protein crystal structure determination.

Streptococcus pneumonia YlxR at 1.35 A shows a putative new fold.

The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 A. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 A from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer alpha/beta sandwich with the overall shape of a cylinder and shows no structural homology to proteins of known structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the alpha-beta plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.

Autotracing of Escherichia coli acetate CoA-transferase alpha-subunit structure using 3.4 A MAD and 1.9 A native data.

The automation of protein structure determination is an essential component for high-throughput structural analysis in protein X-ray crystallography and is a key element in structural genomics. This highly challenging undertaking relies at present on the availability of high-quality native and derivatized protein crystals diffracting to high or moderate resolution, respectively. Obtaining such crystals often requires significant effort. The present study demonstrates that phases obtained at low resolution (>3.0 A) from crystals of SeMet-labeled protein can be successfully used for automated structure determination. The crystal structure of acetate CoA-transferase alpha-subunit was solved using 3.4 A multi-wavelength anomalous dispersion data collected from a crystal containing SeMet-substituted protein and 1.9 A data collected from a native protein crystal.

Using surface-bound rubidium ions for protein phasing.

Rubidium is a monovalent metal that can be used as a counterion in protein solutions. X-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from Thermus thermophilus. Multiple-wavelength anomalous dispersion (MAD) data were collected from a crystal obtained from a solution containing 0.2 M rubidium salt. One molecule of protein (147 amino acids) binds one well ordered and one poorly ordered Rb atom. Phases calculated with the program SHARP were sufficient for automatic tracing and side-chain assignment using the program ARP/wARP. The data show that bound rubidium ions can be used to determine protein structures and to study the interaction of monovalent metal ions with proteins and other macromolecules.

MAD data collection - current trends.

The multiwavelength anomalous dispersion (MAD) method of protein structure determination is becoming a routine technique in protein crystallography. The increased number of wavelength-tuneable synchrotron beamlines capable of performing challenging MAD experiments, coupled with the widespread availability of charge-coupled device (CCD) based X-ray detectors with fast read-out times have brought MAD structure determination to a new exciting level. Ultrafast MAD data collection is now possible and, with the widespread use of selenium in the form of selenomethionine for phase determination, the method is growing in popularity. Recent developments in crystallographic software are complementing the above advances, paving the way for rapid protein structure determination. An overview of a typical MAD experiment is described, with emphasis on the rates and quality of data acquisition now achievable at third-generation synchrotron sources.

Tryptophanase from Escherichia coli: catalytic and spectral properties in water-organic solvents.

In water-methanol and water-dimethylformamide (DMF) (1:1 v/v) solutions tryptophanase from E.coli retains its abilities to form a quinonoid complex with quasisubstrates and to catalyze the decomposition of S-o-nitrophenyl-L-cysteine (SOPC). Both the KM and Vmax values decrease in water-organic media. The affinities of tryptophanase for L-alanine, L-tryptophan, oxindolyl-L-alanine and indole in aqueous methanol are decreased, the effect being stronger for the more hydrophobic substances. In a water solution tryptophanase catalizes the reaction of SOPC with indole to form L-tryptophan while in water-organic solvents only decomposition of SOPC is observed.

2-Methyl-L-tryptophan is a substrate of tryptophanase.

Tryptophanase was generally considered to be inactive towards tryptophan derivatives substituted at 2-position of the indole ring. We have shown that cells containing tryptophanase catalyze the formation of 2-methyl-L-tryptophan from 2-methylindole and L-serine, and from 2-methylindole, pyruvate and ammonium ion. The kinetics of pyruvate formation from 2-methyl-L-tryptophan and its alpha-deuterated analogue catalyzed by homogeneous tryptophanase were examined. The primary deuterium isotope effect (kH/kD = 4.0) as well as the absorption spectrum of tryptophanase complex with 2-methyl-L-tryptophan indicate that the rate of enzymatic reaction of 2-methyl-L-tryptophan is in a considerable degree determined by the stage of removal of alpha-proton.

Tryptophan indole-lyase from Proteus vulgaris: kinetic and spectral properties.

An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites.

Structure of Bacillus subtilis YXKO--a member of the UPF0031 family and a putative kinase.

We determined the 1.6-A resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY-CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus. YXKO homologues are broadly distributed and are by and large described as proteins with unknown function. The YXKO protein has an alpha/beta fold and shows high structural homology to the members of a ribokinase-like superfamily. However, YXKO is the only member of this superfamily known to form tetramers. Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg(2+)-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily. Two adjacent monomers contribute residues to the active site. The crystal structure provides valuable information about the YXKO protein's tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.

Role of arginine 226 in the mechanism of tryptophan indole-lyase from Proteus vulgaris.

In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3;-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 10(5)-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 10(3)-10(4)-fold. The mutant enzyme catalyzed exchange of the C-alpha-proton in complexes with some inhibitors with rates reduced 10(2)-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme-inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme-substrate intermediates.