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. We performed experimental investigation (on rabbits, males, aging three-month) for study of the morpho-functional changes of the pulp tissues with modeling of traumatic pulpitis and direct pulp capping with TS preparation (8 animals, investigated group) and calcium hydroxide (Calasept, NORDISKA DENTAL) preparation (8 animals, comparison group). After 2nd and 6th weeks tissues of tooth were fixed in 10% formalin with performing routine proceeding after decalcification and making histological slides which were investigated. Manifestations of protective adaptive mechanisms have been revealed in the form of inflammatory process two weeks after the injury in the pulp tissue with its resolution six weeks after performing of direct pulp capping with TS with replacement of necrotic area by connective tissue with their delimitation from viable pulp tissue against a background of intensive formation of capillaries. Morphometric study proved dynamical changes of vascular number cross-sections per 1 mm2 from 69.31±4.76 (2 weeks) to 47.38±4.12 (6 weeks) with 49.2±3.47 vascular density in intact group. Cellular density of odontoblasts as changed from 3.92±1.03 x103 per 1 mm2 (2 weeks) to 7.49±1.51 x103 per 1 mm2 (6 weeks) with 8.3±1.02 x103 per 1 mm2 cellular density in intact group. Thus it can be argued that the use of TS as a material for direct pulp capping promotes more active regeneration processes.

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. Two groups of animals were formed (Dutch rabbits, males, aging three-month, weighting 2-2.4 kg). Group of 8 animals with obtained mucosal changes was our comparison group. Other group of 8 animals with obtained mucosal changes was treated by ozone therapy. Histological investigation has been performed. Microscopical examination of tissue had shown that ozone therapy reduces inflammation and edema and is useful in wound healing in soft tissue as disappearance of necrobiotic processes, epithelialization of aphthous defect, growth of akantotic bands, pronounced reducing of inflammatory cells and changing of cellular ratio (with of neutrophils part from 38.30±2.46% to 6.34±0.63%, eosinophils from 5.49±0.23% to 2.87±0.05%), restoration of the cellular layers of the epithelium, moderately pronounced sclerosis of the papillary layer of the lamina propria. Described results allow to conclude that correction of tissual changes in chronic recurrent aphthous stomatitis could be obtained with ozone therapy using.

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. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.

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. In contrast, NepsilonH in histidine H(+)-440 is not aligned for a favourable proton transfer to the pinacolyl O to promote dealkylation, but electrostatic stabilization by histidine H(+)-440 of the developing anion on the phosphonate monoester occurs. Destabilizing interactions between residues and the alkyl fragment of the inhibitor enforce methyl migration from Cbeta to Calpha concerted with C-O bond breaking in soman-inhibited AChE. Tryptophan-84, phenyalanine-331 and glutamic acid-199 are within 3.7-3.9 A (1 A=10(-10) m) from a methyl group in Cbeta, 4.5-5.1 A from Cbeta and 4.8-5.8 A from Calpha, and can better stabilize the developing carbenium ion on Cbeta than on Calpha. The Trp84Ala mutation eliminates interactions between the incipient carbenium ion and the indole ring, but also reduces its interactions with phenylalanine-331 and aspartic acid-72. Tyrosine-130 promotes dealkylation by interacting with the indole ring of tryptophan-84. Glutamic acid-443 can influence the orientation of active-site residues through tyrosine-421, tyrosine-442 and histidine-440 in soman-inhibited AChE, and thus facilitate dealkylation.

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. RESULTS: The persistence of the F-response was decreased significantly in the LMN group only. The F/M amplitude ratio, vibrated-H/rest-H amplitude ratio and conditioned H/rest H amplitude ratio were significantly increased in patients with predominantly UMN, as well as in those with predominantly LMN signs. H/M amplitude ratios did not differ between the various patient groups. CONCLUSION: These findings show that ALS patients, including those without clinical evidence of UMN involvement, have a marked disinhibition of anterior horn motor neurons. The simple tests described could support an UMN abnormality when clinical signs are lacking, and help to establish a diagnosis sooner and more accurately.

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., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

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, 0.15 M NaCl and 2 mM CaCl2 at 7.0+/-0.1 degrees C. The stereoselectivity of the inactivation of factor Xa is 2-10 and favors the levorotatory enantiomers. The pH-dependence of inactivation of factor Xa by (-)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 6.2+/-0.1. Factor Xa reactivates from its phosphonyl adducts through a self-catalyzed intramolecular reaction, which is much influenced by the presence of phospholipids. The rate of reactivation in the absence of phospholipids is not pH dependent at pH <9, but it increases very much at pH >9. In the presence of phospholipids, the pH dependence of the rate constant for reactivation is sigmoidal in the pH 6.5-10.3 range and levels off at pH >9 indicating that the enzyme catalyzes its reactivation. The kinetic pKa for the recovery of factor Xa from its adducts with the PMNs is in the range of 6.7-8.1 and is consistent with the participation of the catalytic His57 in the reactivation process.

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. Strain rate sensitivity of the posterior cruciate ligament was shown in this model, with midsubstance tears occuring in specimens tested at a low rate and avulsion "stripping" injuries from the femoral side occuring at a high rate. A variable pattern of anterior cruciate ligament tears at both high and low rates suggests that the specific injury mechanism may also involve other factors including notch morphology. We present a simplified mathematic model used to estimate posterior cruciate ligament strain during knee hyperextension.

Small molecular products of dealkylation in soman-inhibited electric eel acetylcholinesterase.

Product analysis of dealkylation in P(S)C(S)-soman-inhibited electric eel acetylcholinesterase (AChE) by GC-MS using the selected ion monitoring mode has been carried out. The instrument was calibrated with pure standards of 2,3-dimethyl-1-butene and 2, 3-dimethyl-2-butene in the gas phase and methylene chloride extracts of 2,3-dimethyl-2-butanol and 3,3-dimethyl-2-butanol from the aqueous phase. The dealkylation in soman-inhibited AChE at pH 5.0 +/- 0.2 and 25 degrees C produces close to 40% alkenes and 50-60% 2, 3-dimethyl-2-butanol. No 3,3-dimethyl-2-butanol could be detected to provide direct evidence of the intervention of a secondary carbenium ion in the reaction path. All the products of the reaction originate from a tertiary carbenium ion. These findings are in good agreement with the results of Michel et al. [(1967) Arch. Biochem. Biophys. 121, 29], which were obtained by countercurrent distribution of tritium-labeled products and their identification by scintillation counting. The early experiments were performed with the mixture of the four soman diastereomers, all labeled with tritium in Calpha.

Laser assisted soldering: microdroplet accumulation with a microjet device.

BACKGROUND AND OBJECTIVE: We investigated the feasibility of a microjet to dispense protein solder for laser assisted soldering. STUDY DESIGN: Successive micro solder droplets were deposited on rat dermis and bovine intima specimens. Fixed laser exposure was synchronized with the jetting of each droplet. After photocoagulation, each specimen was cut into two halves at the center of solder coagulum. One half was fixed immediately, while the other half was soaked in phosphate-buffered saline for a designated hydration period before fixation (1 hour, 1, 2, and 7 days). After each hydration period, all tissue specimens were prepared for scanning electron microscopy (SEM). RESULTS: Stable solder coagulum was created by successive photocoagulation of microdroplets even after the soldered tissue exposed to 1 week of hydration. CONCLUSIONS: This preliminary study suggested that tissue soldering with successive microdroplets is feasible even with fixed laser parameters without active feedback control.

The pH dependence of dealkylation in soman-inhibited cholinesterases and their mutants: further evidence for a push-pull mechanism.

Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.

Laser assisted soldering: effects of hydration on solder-tissue adhesion.

Wound stabilization is critical in early wound healing. Other than superficial skin wounds, most tissue repair is exposed to a hydrated environment postoperatively. To simulate the stability of laser-soldered tissue in a wet environment, we studied the effects of hydration on laser soldered rat dermis and baboon articular cartilage. In this in vitro study, we used a solder composed of human serum albumin, sodium hyaluronate, and Indocyanine Green. A 2 µL solder droplet was deposited on each tissue specimen and then the solder was irradiated with a scanning laser beam (808 nm and 27 W/cm2). After photocoagulation, each tissue specimen was cut into two halves dividing the solder. One half was reserved as control while the other half was soaked in saline for a designated period before fixation (1 h, 1, 2, and 7 days). All tissue specimens were prepared for scanning electron microscopy (SEM). SEM examinations revealed nonuniform coagulation across the solder thickness for most of the specimens, likely a result of the temperature gradient generated by laser heating. Closer to the laser beam, the uppermost region of the solder formed a dense coagulum. The solder aggregated into small globules in the region anterior to the solder-tissue interface. All cartilage specimens soaked in saline suffered coagulum detachment from tissue surface. We noted a high concentration of the protein globules in the detached coagulum. These globules were likely responsible for solder detachment from the cartilage surface. Solder adhered better to the dermis than to cartilage. The dermal layer of the skin, composed of collagen matrix, provided a better entrapment of the solder than the smooth surface of articular cartilage. Insufficient laser heating of solder formed protein globules. Unstable solder-tissue fusion was likely a result of these globules being detached from tissue substrate when the specimen was submerged in a hydrated environment. The solder-tissue bonding was compromised as a result of this phenomenon. © 1998 Society of Photo-Optical Instrumentation Engineers.

Modulation of human alpha-thrombin activity with phosphonate ester inhibitors.

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH3; and CH3O) inactivate human alpha-thrombin with rate constants 4-235 M-1 s-1 in pH 6.5, 0.025 M citrate buffer, and 0.15 M NaCl at 7.0 +/- 0.1 degrees C. Stereoselectivity of the inactivation of thrombin is 2-39 and favors the levorotatory enantiomers. The pH-dependence of inactivation of thrombin by (-)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 8.0 +/- 0.1 in 0.15 M NaCl and a pKa of 7.4 +/- 0.2 in 0.15 M choline chloride in the nucleophilic attack of the catalytic Ser at phosphorus. The solvent isotope effect on ki/Ki in the pH-independent region of the reaction is 2.26 +/- 0.17. Thrombin activity returns from the adducts on the 2-7 h time scale at 25.0 +/- 0.1 degrees C via a self-catalyzed intramolecular reaction. The pH dependence of reactivation is significant from the adduct formed with (-)-CH3O-PMN and (-)-CH3-PMN and less so from the adducts formed with the other enantiomers of the inhibitors. Kinetic pKs approximately 7.2, with the exception of the adducts with (-)-PMN and (-)-CH3O-PMN, indicate that a pH-dependent conformational change affects the rate of dephosphonylation. A structural interpretation of the stereoselectivity and other mechanistic features is provided based on the energy-optimized structures of the adducts. Pharmaco-medical use of human alpha-thrombin covalently modified by the PMNs is suggested.

Unique push-pull mechanism of dealkylation in soman-inhibited cholinesterases.

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(-)C(+) and P(-)C(-) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 +/- 0.2 degrees C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4-4.6 and pK2 6.3-6.5 for Ee AChE and pK1 4.8-5. 0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13-16 x 10(-3) s-1 for Ee AChE and 8 x 10(-3) s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1-1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 +/- 0.2 degrees C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 +/- 0.2 degrees C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 +/- 0.2 degrees C are pH independent and approximately 10(4) times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >10(10) times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cbeta to Calpha. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C-O bond breaking. The positive charge at the rate-determining transition state is distributed between Cbeta and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.

Small-angle HeNe laser light scatter and the compressive modulus of articular cartilage.

Light scattering is a widely used technique for probing the microarchitecture and interactions of biological materials and solutions. In this paper, we describe the use of this method in the study of articular cartilage. The experiments presented utilize small-angle static scattering of HeNe laser light (632.8 nm) from 40 microns thick samples of cartilage taken from the superficial zone of baboon femoral condyle. The specimens were taken from a total of 26 sites in eight animals of various ages. In addition to measuring the dependence of the intensity of scattered light on scatter angle, we performed mechanical testing at the test sites using creep-indentation techniques. The results from the optical and mechanical experiments were compared, and a significant correlation was noted between the average scatter angle and the compressive aggregate modulus. In addition, it was noted that the cartilage of skeletally immature animals had a smaller aggregate modulus and scattered to a higher average angle than the cartilage of skeletally mature animals. A quantitative theory was developed to explain the relation, between mechanical and optical properties in terms of the degree of order in the spatial arrangement of the collagen fibers in cartilage.

Reversible modification of tissue-type plasminogen activator by methylphosphonate esters.

In spite of their rapid aqueous hydrolysis, 4-nitrophenyl 4-X-phenacyl methylphosphonates (X = H, (PMN) CH3, CH3O, Cl and NO2) inactivate many serine proteases of the pancreatic and blood coagulation systems efficiently. The rate constants, K/Ki, for the inactivation of tissue-type plasminogen activator enzyme (t-PA) are 470-750 M-1 S-1 with PMN, 4-CH3-PMN, and 4-CH3O-PMN in pH 7.8, 0.05 M Tris buffer at 7.0 +/- 0.5 degrees C, but t-PA cannot be inhibited with the 4-Cl and NO2 derivatives due to rapid competing hydrolysis. Enzyme activity returns from each enzyme-adduct at a characteristic rate, due to a self-catalyzed intramolecular reactivation process. The rate constants for spontaneous reactivation of t-PA from the adducts formed with the three inhibitors are K = 0.25-12.3 x 10(-2) min-1 at pH 7.4 and 25.0 +/- 0.1 degrees C and pH-dependent with an apparent pK approximately 8.3. The recovery of t-PA activity from the adducts in 40% human plasma buffered at pH 7.4 is the same or twice that in plain buffer. The presence of fibrin has a slight effect on inactivation but not on reactivation. The modulation of enzyme activity by reversible generation of the phosphonylated adducts has potential for medical application.

A molecular theory of cartilage viscoelasticity.

Recent work on the subject of cartilage mechanics has begun to focus on the relationship between the microscopic structure of cartilage and its macroscopic mechanical properties (Bader et al., Biochem. Biophys. Acta, 1116 (1992) 147-154; Buschmann, PhD Thesis, Massachusetts Institute of Technology, 1992; Kovach, Biophys. Chem., 53 (1995) 181-187; Lai et al., J. Biochem. Eng., 113 (1991) 245-248; Armstrong and Mow, J. Bone Jt. Surg., 64A (1982) 88; Jackson and James, Biorheology, 19 (1982) 317-330). This paper reviews recent theoretical developments and presents a comprehensive explanation of the viscoelastic properties of cartilage in terms of molecular structure. In doing this, a closed form hybrid solution to the non-linear, cylindrical Poisson-Boltzmann equation is developed to describe the charge-dependent component of the equilibrium elasticity arising from polysaccharide charge (Benham, J. Chem. Phys., 79 (4) (1983) 1969-1973; Einevoll and Hemmer, J. Phys. Chem., 89 (1) (1988) 474-484; Fixman, J. Chem. Phys., 70 (11) (1979) 4995-5001; Ramanathan and Woodburg, J. Chem. Phys., 82 (3) (1985) 1482-1491; Wennerstrom et al., J. Chem. Phys., 76 (9) (1982) 4665-4670). This solution agrees with numerical solutions found in the literature (Buschmann, PhD Thesis, Massachusetts Institute of Technology, 1992). The charge-independent, entropic contribution to the equilibrium elasticity is explained in a manner similar to that recently presented for concentrated proteoglycan solution (Kovach, Biophys. Chem., 53 (1995) 181-187). This approach exploits a lattice model of the solution, subject to a Bragg-Williams type approximation to derive the volume dependence of polysaccharide configuration entropy (Flory, Principles of Polymer Chemistry, Cornell University Press, Ithaca, NY, 1953; Huggins, Some properties of Solutions of Long-chain Compounds, 1941, pp. 151-157; Stanley, Introduction to Phase Transitions and Critical Phenomena, Oxford University Press, Oxford, 1971). Together, these two contributions accurately reproduce the experimentally determined osmotic pressure of cartilage as previously determined by Maroudas (Maroudas and Bannon, Biorheology, 18 (1981) 619-632). The time-dependent, or creep, phenomena which cartilage exhibits when subject to mechanical load is explained in terms of frictional drag on the polysaccharide chain monomers in terms of a Kirkwood-Riseman type model (Kirkwood and Riseman, J. Chem. Phys., 16 (6) (1948) 573-579). This approach is shown to accurately predict the hydraulic permeability of cartilage as previously determined by Maroudas (Madouras, Ann. Rheum. Dis., 34 (suppl. 3) (1975) 77). By use of a quasi-static approximation (neglecting inertial effects) the time-dependent response to a uniform compressive force is determined and also found to be in good agreement with experimental values from the literature.

Origins and diversity of the aging reaction in phosphonate adducts of serine hydrolase enzymes: what characteristics of the active site do they probe?

Molecular mechanics and dynamics combined with semiempirical calculations were carried out for purposes of comparison of the active site characteristics of AChE, trypsin, and chymotrypsin as probed by their diastereomeric adducts with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman), methylphosphonate monoester anions, and tetravalent carbonyl intermediates of the reactions of the natural substrates in each case. Glu199 is a key residue in the electrostatic catalytic mechanism of AChE, in removal of the leaving group, and possibly by acting as an alternate general base catalyst. "Pushing" of an alkoxy ligand by Glu199 and the numerous small van der Waals interactions promote dealkylation in phosphonate adducts of AChE much more effectively than any other enzyme. A high concentration of negative charge created by the phosphonate ester monoanion and Glu199 adjacent to it fully accounts for the resistance to the attack of even the strongest nucleophile applied for enzyme reactivation. Stabilization of the developing negative charge on the phosphonates in the soman-inhibited PSCS adducts of serine hydrolases is by electrophilic residues in the oxyanion hole (AChE) and the protonated catalytic His. PR diastereomers of soman-inhibited AChE can be accommodated in an orientation in which the oxyanion hole interactions are lost and for which the stabilizing interactions are 17-26 kcal/mol smaller than in the PS diastereomer. The dealkylation reaction is almost equally likely in all diastereomers of soman-inhibited AChE. The stabilizing interaction energies are approximately 4 kcal/mol greater in the PR than in the PS adducts of the soman-inhibited serine proteases. There is 0.60 unit greater partial negative charge on the phosphonyl fragment in the anion of phosphonate monoesters of Ser than at the oxygens of tetravalent carbonyl transients resulting in approximately 12-22 kcal/mol greater stabilization of the former than the latter.