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PURPOSE: The impact of nuclear reaction model differences on simulation of prompt gamma-ray imaging for proton therapy range verification was assessed. Four nuclear reaction models were used to simulate gamma emission in proton beams, and were validated against experimental cross-sections. METHODS: Proton-induced nuclear reactions on carbon, oxygen, nitrogen and calcium were investigated with the Monte Carlo toolkits GEANT4 9.5 and MCNPX 2.7, and the dedicated nuclear reaction codes TALYS 1.4 and EMPIRE 3.1. Absolute cross-sections of discrete prompt gamma lines and the total gamma production were obtained for the 1-200 MeV incident proton energy range. They were compared to 34 discrete line measurements reported in literature. Using these cross-sections, we analyzed the gamma production along the path of proton beams passing through various tissues. RESULTS: The differences in absolute discrete line cross-sections as predicted by the models ranged from almost zero to an order of magnitude, depending on the gamma line and incident proton energy. Overall, the dedicated nuclear reaction codes provided a better fit to most experimental excitation functions. For a 150 MeV proton beam stopping in soft tissue, these differences amount to a variation by a factor of 4 of the gamma emission around the Bragg peak location. The maximum of gamma production near the end of proton range differed by 7 mm, and the change of the 50% emission fall-off position was 4 mm. CONCLUSIONS: There is a clear need for improvement of nuclear reaction models to accurately simulate proton range verification using prompt gamma-rays. Current simulation codes show large uncertainties in both the total gamma yield and the correlation of gamma emission with the proton Bragg peak. GEANT4 and MCNPX in particular appear to have limited predictive power.

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PURPOSE: To investigate the necessity of the verification of dose distributions using Monte Carlo (MC) simulations for proton therapy of head and neck patients and other complex patient geometries. METHODS: TOPAS, a TOol for PArticle Simulations that makes MC simulations easy-to-use for research and clinical use and is layered on top of Geant4, has been used to simulate the treatments of head and neck patients at the Massachusetts General Hospital (MGH). The resulting dose distributions have been compared to pencil beam calculations based on the XiO treatment planning system. Dose difference distributions were used to highlight areas where the two algorithms did not agree. Dose volume histograms are utilized to investigate the overall agreement of the planned doses in target structures. RESULTS: 21 head and neck patients, both nasopharynx and spinal cord, were investigated. The field complexity ranges from a single field up to 13 fields. For all patients, the dose in the clinical target volume agrees well. Nevertheless, differences in critical structures around the targets have been observed mostly due to range differences between the two algorithms. CONCLUSIONS: Pencil beam algorithms provide an accurate description of dose in the target volume. However, we conclude that the differences between MC simulations and pencil beam algorithms in regions outside the target for complex geometries, such as present in head and neck patients, support the necessity of routine use of MC simulations for treatment verifications before treatment. TOPAS is aiming to make such routine simulations available to all researchers and clinics. An automated interface utilizing TOPAS to enable such simulations has been developed at MGH and should become routinely used in the near future for patients with complex geometries. NIH/NCI R01 CA140735.

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OBJECTIVE: To investigate trends in socio-economic inequalities in smoking, alcohol and drug use in the community of Utrecht, the Netherlands. DESIGN: Repeated cross-sectional population surveys. METHOD: Questionnaires were used to collect information about smoking, (problematic) alcohol use and the use of cannabis, ecstasy and other drugs in the general population aged 15-64 years in Utrecht, the Netherlands. Data were collected in 1999 on 2485 responders (response: 56%) and in 2003 on 1840 responders (54%). The male-female ratio was 4:6. RESULTS: The percentage of smokers was 43 in 1999 and 34 in 2003. Excessive use of alcohol was reported by 21% in both 1999 and 2003, cannabis use was reported in 13% and 14%, respectively, and use of hard drugs was reported in 1-3% in both 1999 and 2003. Native Dutch people, men, unmarried people and people with a low education or income level were at increased risk for drinking, smoking and using drugs. People with a low socio-economic status were just as likely to smoke in 1999 as in 2003, while the use of alcohol, cannabis and ecstasy increased. Those with a higher socio-economic status were much less likely to smoke in 2003 than in 1999, while fewer used cannabis and ecstasy use remained the same. CONCLUSION: Socio-economic inequalities in substance abuse were greater in 2003 than in 1999 in the studied population in Utrecht. People with a low socio-economic status showed an increase in unhealthy behaviour concerning the use of alcohol, cannabis and ecstasy.

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Using the catalytic mechanism of lysozyme as a paradigm for the mechanism of other enzymes that catalyze the hydrolysis of beta-1,4-glycosidic linkages, including chitinase, we have examined the effect of chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) on the reaction catalyzed by Zea mays chitinase. Inactivation with EDC did not result in derivatization of essential carboxylic acid residues, but resulted in the selective modification of a single essential tyrosine residue (Verburg, J. G., Smith, C. E., Lisek, C. A., and Huynh, Q. K., 1991, J. Biol. Chem. 267, 3886-3893). Here, we examine the role of the homologous tyrosine residue in the catalytic mechanism of the Arabidopsis thaliana chitinase. Tyrosine-174 of the Arabidopsis chitinase was replaced, with phenylalanine, alanine, histidine, and methionine by site-directed mutagenesis, and the variant chitinases were expressed in insect cells using baculovirus transfer vectors. A comparison of the reaction catalyzed by each of the variant enzymes indicates that substitution of another amino acid for Tyr-174 alters, but does not eliminate, enzymatic activity. Estimates of the specific activities of the variant chitinases reveal that substitution of His for Tyr-174 has a minimal effect on catalysis, the specific activities of the Phe and Met variants are approximately equivalent to each other, but are 60% the specific activity of wild-type Arabidopsis chitinase, and the specific activity of the Ala variant is only 40% that of wild-type. The observation that the Arabidopsis chitinase is tolerant to mutagenesis at this position suggests that Tyr-174 does not participate directly in catalysis.

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Sodium pentobarbital (Nembutal) is often used to block the pro-oestrous luteinizing hormone (LH) surge in rats. Nembutal is also known to lower body temperature. This study was designed to investigate whether Nembutal affected the time course of meiosis and timing of ovulation induced by exogenous hormones, and whether the possible effects of Nembutal on these processes were related to temperature. Gonadotrophin-releasing hormone (GnRH), the GnRH-analogue Ovalyse, or rat luteinizing hormone (LH) were administered to trigger resumption of meiosis and ovulation; Nembutal (35 mg kg-1 body weight) or saline was given 10 or 60 min later. Plasma profiles of LH were measured and Graafian follicles were studied histologically for meiotic progress and ovulation. Nembutal suppressed the spontaneous surge of LH at pro-oestrus and caused a long-lasting decrease in body temperature. If 1000 ng GnRH was given 2 h before the pro-oestrous LH surge, most of the oocytes had extruded a polar body 10 h later and most follicles had ovulated 14 h later. Nembutal given 1 h after GnRH delayed extrusion of the polar body and ovulation by about 2 h. Nembutal caused a similar delay in ovulation when it was administered after 100 ng of Ovalyse, and it also delayed meiosis when given after 1000 ng of LH. This effect of Nembutal was prevented if body temperature was maintained at 37 degrees C. The delaying effect of Nembutal on meiosis and ovulation induced by exogenous GnRH or LH is related to a long-lasting decrease in body temperature.

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Chitinase isolated from Zea mays seeds is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the absence of exogenous nucleophiles. Oligomers of N-acetylglucosamine,N,N',N",N"'-tetra-N-acetylchitotetraose (GlcNAc4), and to a lesser extent, N,N',N"-tri-N-acetylchitotriose (GlcNAc3) and N,N'-di-N-acetylchitobiose (GlcNAc2) provide partial protection against inactivation by the reagent. An examination of the concentration dependence of the protection afforded by GlcNAc4 revealed direct competition between the substrate analog and the reagent for the same binding sites on the enzyme. Isolation and Edman degradation of a "new" tryptic fragment, observed after inactivation of chitinase with EDC, revealed the sequence G-P-L-Q-I-S-W-N-*-N-Y-G-P-A-G-R, where the asterisk represents a cycle in which no amino acid was detected, presumably as a consequence of derivatization with EDC. In basic chitinases from dicotyledonous plants such as Arabidopsis thaliana, Phaseolis vulgaris (bean), Nicotiana tabacum (tobacco), and Solanum tuberosum (potato), as well as in the chitinase isolated from the monocotyledonous plant Hordeum vulgare (barley), this position is invariably occupied by a tyrosine. However, in the Oryza sativa (rice) basic chitinase, this position is occupied by a phenylalanine. The following additional evidence supports identification of this residue as tyrosine in Z. mays chitinase. (a) Inactivation of chitinase with EDC is reversible by treatment with hydroxylamine. (b) Liquid secondary ion mass spectrometric analysis of the isolated derivatized peptide revealed the presence of a molecular ion with a mass to charge ratio consistent with the peptide containing a derivatized tyrosine residue. These results provide evidence for an essential tyrosine residue at or near the catalytic site of chitinase that is selectively modified during inactivation with EDC.

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Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907-914).

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The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.

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This study employs classical inviscid fluid dynamics theory to investigate whether LV diastolic inflow volume and the size of the LV play a role in vortex ring formation. Fluid injection across an orifice into a large container results in the generation of a vortex ring having a constant size and speed. Relations between the vortex size and speed and the injection were obtained by applying conservation laws regarding kinetic energy, impulse and vorticity; the initial state was computed using a bolus injection model, and the final state by using the Kelvin vortex model. An important parameter in the equations is the relative injection length, i.e., the ratio of the length of the injected bolus and the radius of the orifice (L/R). Its estimated highest value in man, L/R = 15, produces a rather thick vortex ring (relative thickness 0.77). Comparable results following from the Hill vortex model convinced us that the Kelvin vortex model can be applied in the whole range of injection lengths in the human left ventricle. In an in vitro model it is shown experimentally that vortex rings can be generated for L/R in the range from 2 to 16. The measured traveling speed of the vortex ring is in fair agreement with the theory, as well as the ring radius for large injections. A vortex ring located in a narrow channel cannot reach its proper traveling speed. The method of images is used to estimate the speed reduction of vortex rings within a cylinder. It turns out that propagation of vortex rings is possible when the ratio of orifice to cylinder radius is less than about 0.5.(ABSTRACT TRUNCATED AT 250 WORDS)

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The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.

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The aziridinium of purified quinacrine mustard at 50 microM inactivates the bovine heart mitochondrial F1-ATPase with a pseudo-first order rate constant of 0.07 min-1 at pH 7.0 and 23 degrees C. An apparent Kd of 27 microM for the enzyme-reagent complex was estimated from the dependence of the rate of inactivation on the concentration of quinacrine mustard. The pH inactivation profile revealed that deprotonation of a group with a pKa of about 6.7 is necessary for inactivation. The amount of reagent incorporated into the protein increased linearly with the extent of inactivation. Complete inactivation was estimated to occur when 3 mol of reagent were incorporated/mol of F1. Enzyme, in which steady state ATPase was inactivated by 98% by quinacrine mustard, hydrolyzed substoichiometric ATP with zero order kinetics suggesting that residual activity is catalyzed by F1 in which at least one beta subunit is modified. By exploiting the reactivity of the aziridinium of covalently attached reagent with [3H] aniline, sites modified by quinacrine mustard were labeled with 3H. Isolation of radioactive cyanogen bromide peptides derived from F1 inactivated with the reagent in the presence of [3H]aniline which were identified by sequence analysis and sequence analyses of radioactive tryptic fragments arising from them have revealed the following. About two thirds of the radioactivity incorporated into the enzyme during inactivation is apparently esterified to one or more of the carboxylic acid side chains in a CNBr-tryptic fragment of the beta subunit with the sequence: 394DELSEEDK401. The remainder of the radioactivity is associated with at least two sites within the cyanogen bromide peptide containing residues 293-358 of the beta subunit. From these results it is concluded that inactivation of F1 by the aziridinium of quinacrine mustard is due, at least in part, to modification of one or more of the carboxylic acid side chains in the DELSEED segment of the beta subunit and possibly also to modification of unspecified amino acid side chains between residues 302-356 of the beta subunit.

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To determine whether duodenogastric reflux into the thoracic stomach could be caused by the transmission of negative intrapleural pressure fluctuations into the gastric lumen, a physical model is described and an equation calculated Pm + Pa - Pmb - (Sv.Pmb.Vmb/Pm) = Ppl - Sv.Vmb where Pm is intragastric pressure, Pa is atmospheric pressure, Pmb is end-expiratory gastric base pressure, Vmb is corresponding gastric volume, Sv is stiffness of gastric wall, and Ppl is intrapleural pressure. The validity of the model is demonstrated in six anesthetized mongrel dogs (18-31 kg) in which a thoracic stomach was constructed. The transmission of the intrapleural pressure fluctuations across the gastric wall proved to be greatly influenced by the gastric stiffness. The latter parameter varied from 0.05 to 1.97 cmH2O/ml, corresponding with a pressure transmission of 100 and 60%, respectively. Because high degrees of gastric stiffness are only present for large gastric volumes or when gastric peristalsis is present, it is concluded that, in general, the intrapleural pressure fluctuations are transmitted into the thoracic stomach. For this reason, respiratory efforts may play an important role in inducing duodenogastric reflux into the thoracic stomach.

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The characteristics of ATP hydrolysis at a single catalytic site of the bovine heart F1-ATPase (MF1) as originally described by Grubmeyer et al. (Grubmeyer, C., Cross, R.L., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100) were compared with those of various chemically modified preparations of MF1 in which the steady state activity was severely attenuated. Although it was not necessary to age our preparations of native MF1 in the presence of 2 mM Pi to observe the same characteristics of single site catalysis, such aging did shift the equilibrium of bound substrate and bound products at the single catalytic site in favor of ATP. After loading a single catalytic site on the enzyme with substoichiometric [alpha,gamma-32P]ATP, the addition of 5-20 microM ATP or ADP was effective in promoting both the hydrolysis of bound [alpha,gamma-32P]ATP and release of radioactive products. Under these conditions, the 5-20 microM ATP added as promoter was hydrolyzed at a rate commensurate with the turnover rate of the enzyme, whereas the promoted hydrolysis of the [alpha,gamma-32P]ATP, preloaded at a single catalytic site, was considerably slower. Therefore, the high affinity, single catalytic site loaded first does not directly contribute to steady state ATP hydrolysis. That the single, high affinity catalytic site is not a "normal" catalytic site is supported by the properties of enzyme modified by 5'-p-fluorosulfonylbenzoyladenosine which exhibits only slightly altered characteristics of single site catalysis and promoted single site catalysis, despite exhibiting severely attenuated steady state turnover. Other modified forms of the enzyme in which the steady state activity was severely attenuated by derivatization with 5'-p-fluorosulfonylbenzoylinosine, 7-chloro-4-nitrobenzofurazan, or 1,5-difluoro-2,4-dinitrobenzene also bound substoichiometric ATP at a single catalytic site. However, the characteristics of single site hydrolysis by these modified forms of the enzyme differed considerably from those of native MF1.

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From knee extension moments measured with a dynamometer, the quadriceps muscle force, the patellar ligament force and the reaction force in the patellofemoral joint at various knee angles (0-90 degrees) were estimated. The information needed to calculate the combined effect of both patellofemoral and tibiofemoral joint on the mechanical advantage of the muscle was obtained from lateral-view radiographs of autopsy knees. The results show that the smallest quadriceps force (2,000 N) is exerted at maximal extension, and the largest force (8,000 N) at about 75 degrees of flexion. The patellar ligament force reaches a maximum (5,000 N) at 60 degrees. The reaction force in the patellofemoral joint is the smallest (1,000 N) at extension and is of the same values as the muscle force in a range from 75 to 90 degrees. Especially at large flexion angles, the value of the estimated forces is considerably larger (by 100%) than reported in the literature. This difference is attributed to the influence of the patellofemoral joint on the mechanical advantage of the muscle, which has not been taken into account in other studies.

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When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.4, 1.4 mol of [14C]Nbf were incorporated per mol of enzyme. After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable. This radioactive peak, designated RP-1, eluted with a retention time of 95 min. When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min. Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl [14C]Nbf-O-tyrosine. These results provide the basis for a facile method to purify peptides containing [14C]Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.

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A mathematical model of the patellofemoral joint taking into account movements and forces in the sagittal plane is described. The system parameters of the model are the locations of the attachments of the quadriceps muscle and the patellar ligament, the length of the patellar ligament, the dimensions of the patella and the geometry of the articulating surfaces. They were obtained from ten autopsy knees. The model enables calculation of the relative position of the patella, patellar ligament and quadriceps tendon, the location of the patellofemoral contact point and the magnitude of the patellofemoral compression force and the force in the patellar ligament as a function of the location of the tibial tuberosity at different flexion-extension angles of the knee. The model is validated by comparing model data with experimentally determined data.

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