Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System.

Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C-catalyzed reactions revealed that specific binding of effectors at S, n = 1-4 and S', n = 1-3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin.

Predicting Significant Iliac Vein Compression Using a Probability Scoring System Derived From Minimal Luminal Area on Computed Tomography Angiography in Patients 65 Years of Age or Younger.

Background: The presence of 50% or more stenosis on intravascular ultrasound (IVUS) is considered diagnostic of iliac vein compression (ILVC) by most operators. We have previously developed a scoring system combining minimal luminal area (MLA) at the compression site and age to predict ILVC as seen on IVUS. We present a revised and improved scoring system following an additional number of patients and limited to patients 65 years of age and younger.

Features of the morphological state of bone tissue of the lower wall of the maxillary sinus with the use of fixed orthodontic appliances.

Aim: The aim of the study was to identify the consequences of the use of fixed otrhodontic appliances (FOAs) on the morphological state of the tissues of the lower wall of the maxillary sinus, depending on the duration of their use.

Materials And Methods: The study involved 115 male and female patients aged 18 to 25 years. All patients were divided into 2 research groups depending on the period of wearing brace systems.

INFLUENCE OF TRICALCIUM SILICATE ON COURSE OF TRAUMATIC PULPITIS.

The use of Tricalcium Silicate (TS) as an odontotropic preparation makes it possible to create a hermetic crown restoration with a high degree of adhesion. However, the use of TS silicate by direct pulp capping remains disputable. The aim of this study was to determine the effects of TS on course of traumatic pulpitis by detection of morpho-functional peculiarities of changes in pulp tissue.

INFLUENCE OF OZONE THERAPY ON ORAL TISSUE IN MODELING OF CHRONIC RECURRENT APHTHOUS STOMATITIS.

Chronic recurrent aphthous stomatitis (CRAS) belongs to the group of chronic, inflammatory, ulcerative diseases of the oral mucosa. The aim of this study was to determine the effects of ozone on the morphofunctional peculiarities of the soft tissues in modeling chronic recurrent aphthous stomatitis. We performed experimental investigation for study of the morpho-functional state of tissues of the oral mucosa in CRAS with using of previously proposed and widely used modeling scheme with ovalbumin and aluminum hydroxide.

Janus emulsion mediated porous scaffold bio-fabrication.

A three dimensional biopolymer network structure with incorporated nano-porous calcium phosphate (CaP) balls was fabricated by using gelatin-chitosan (GC) polymer blend and GC stabilized olive/silicone oil Janus emulsions, respectively. The emulsions were freeze-dried, and the oil droplets were washed out in order to prepare porous scaffolds with larger surface area. The morphology, pore size, chemical composition, thermal and swelling behavior was studied by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and micro-Differential Scanning Calorimetry (micro-DSC).

Nano-porous calcium phosphate balls.

By dropping a NaH2PO4·H2O precursor solution to a CaCl2 solution at 90°C under continuous stirring in presence of two biopolymers, i.e. gelatin (G) and chitosan (C), supramolecular calcium phosphate (CP) card house structures are formed.

Equilibrium topology and partial inversion of Janus drops: a numerical analysis.

The equilibrium topology of an aqueous Janus emulsion of two oils, O1 and O2, with water, W, [(O1+O2)/W], is numerically evaluated with the following realistic interfacial tensions (γ): γO2/W =5 mN m(-1) , γO1/O2 =1 mN m(-1) , and γO1/W varies within the range 4-5 mN m(-1) , which is the limiting range for stable Janus drop topology. The relative significance of the two independently pivotal factors for the topology is evaluated, that is, the local equilibrium at the line of contact between the three liquids and the volume fraction of the two dispersed liquids within the drop. The results reveal a dominant effect of the local equilibrium on the fraction of the O2 drop surface that is covered by O1.

The deviant ATP-binding site of the multidrug efflux pump Pdr5 plays an active role in the transport cycle.

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance.

Proton bridging in the interactions of thrombin with hirudin and its mimics.

Thrombin is the pivotal serine protease enzyme in the blood cascade system and thus a target of drug design for control of its activity. The most efficient nonphysiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging, and hydrophobic interactions with thrombin to achieve tight binding with K(i) values in the nano- to femtomolar range.

Proton bridging in the interactions of thrombin with small inhibitors.

Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin.

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT.

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.

Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.

Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195 OgammaH and His57 Nepsilon2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with fractionation factors 0.66 +/- 0.03 under enzyme saturation with substrate and 0.

Proton inventory studies of alpha-thrombin-catalyzed reactions of substrates with selected P and P' sites.

Deuterium kinetic solvent isotope effects for the human alpha-thrombin-catalyzed hydrolysis of (1) substrates with selected P(1)-P(3) sites, Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7-AMC, Bz-Phe-Val-Arg-4-nitroanilide (pNA), and H-D-Phe-L-Pip-Arg-pNA, are (DOD)k(cat) = (2.8-3.3) +/- 0.

Early detection of hepatic encephalopathy by recording visual evoked potential (VEP).

The visual evoked potential (VEP) record in response to a pattern stimulus is a non invasive and reliable method of detecting central and peripheral nerve system abnormalities. VEP recording have been used in animals with fulminant hepatic failure, and also in-patients with hepatic encephalopathy and acute severe hepatitis. Our aims were: a.

Effects of femoral neck length, stem size, and body weight on strains in the proximal cement mantle.

Background: Several studies have shown that certain cemented total hip replacement femoral stems have been associated with the complications of early debonding, loosening, and osteolysis. Some authors have suggested that these failures may be related to the surface finish of the stems. We developed an in vitro biomechanical experiment characterized by simulated stair-climbing to investigate the multiple factors involved in loosening of cemented femoral stems.

Electrophysiologic evaluation of upper motor neuron involvement in amyotrophic lateral sclerosis.

Background: The demonstration of upper motor neuron (UMN) dysfunction in patients with amyotrophic lateral sclerosis (ALS) with predominantly lower motor neuron (LMN) signs is clinically sometimes difficult.

Methods: We analyzed the possible role of F and H waves in the diagnosis of UMN dysfunction in 36 patients with different clinical forms of ALS and 20 controls. In both lower limbs we evaluated F-wave persistence and the F/M ratio of tibial nerves, Hmax/Mmax ratio, vibratory and recurrent (paired) inhibition of the H-reflex.

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies.

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al.

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants.

The role of active-site residues in the dealkylation reaction in the P(S)C(S) diastereomer of 2-(3,3-dimethylbutyl)methylphosphonofluoridate (soman)-inhibited Torpedo californica acetylcholinesterase (AChE) was investigated by full-scale molecular dynamics simulations using CHARMM: >400 ps equilibration was followed by 150-200 ps production runs with the fully solvated tetracoordinate phosphonate adduct of the wild-type, Trp84Ala and Gly199Gln mutants of AChE. Parallel simulations were carried out with the tetrahedral intermediate formed between serine-200 Ogamma of AChE and acetylcholine. We found that the NepsilonH in histidine H(+)-440 is positioned to protonate the oxygen in choline and thus promote its departure.

NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase.

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S.

Reversible modulation of human factor Xa activity with phosphonate esters: media effects.

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH3 and CH3O) inactivate human factor Xa with rate constants 8-86 M(-1)s(-1) at pH 6.75 in 0.025 M Hepes buffer, 0.

Cruciate injury patterns in knee hyperextension: a cadaveric model.

We created an experimental model to evaluate the effects of strain rate on the mechanism of combined cruciate ligament injuries in knee hyperextension. Using straight knee hyperextension to rupture the anterior and posterior cruciates, two strain rates (approximately 100% per second and 5400% per second) were applied to reproduce two clinical injury patterns of the knee: low energy (sporting) and high energy (pedestrian-motor vehicle accident). Ten pairs of fresh-frozen cadaveric knees were injured to 45 degrees of hyperextension.