Safe pseudoelastic limit range under torsional loading with Reciproc Blue.

AIM: To determine the distortional angle and torsional load at the pseudoelastic limit of Reciproc Blue instruments and to verify the safety of using pre-set dedicated motors designed for use with the original Reciproc instruments. METHODOLOGY: Two torsional conditions of Reciproc R25 and Reciproc Blue R25 were tested using a custom device. The first condition fixed the file tips at 3 mm and repetitively rotated them, with gradually increasing angles, from 10° to 270°, and the resulting torque was recorded (n = 15). The second test involved a single continuous rotation until fracture (n = 15). The pseudoelastic limits of the instruments were determined from their torque-rotation curves. For statistical analysis, two-way analysis of variance and t-tests were used, at a 95% significance level. Tested specimens were examined using field-emission scanning electron microscopy (FE-SEM). RESULTS: The angle at the pseudoelastic limit was significantly greater for Reciproc Blue than Reciproc, regardless of test mode (P < 0.05). When torsional loading was repeated, using gradually increasing rotational angles, the torsional resistance was significantly lower than for a single rotation (P < 0.05). However, under all test conditions, the pseudoelastic limit was below the pre-set 170° of the dedicated reciprocating motor. FE-SEM evaluation of the lateral aspects of the instruments revealed numerous longitudinal microcracks running along their long axis. For Reciproc Blue groups, the file shaft machining grooves were distorted after repetitive and continuous torsional tests. CONCLUSIONS: The 170° pre-set angle of the dedicated endodontic motors for the Reciproc system was safe for Reciproc Blue in single or time-restricted use.

Anatomical relationship of maxillary posterior teeth with the sinus floor and buccal cortex.

This study aimed to evaluate the possibility of root fenestration or oroantral communication by evaluating the distance from root apex to the sinus floor and buccal cortex in maxillary posterior teeth using cone-beam computed tomography (CBCT) images. The study included 2182 roots of the maxillary posterior teeth from 219 patients after reviewing CBCT images of 462 patients according to the location of roots by two endodontists. The distances from each root apex to the maxillary sinus floor and buccal and palatal cortices were evaluated according to sex and age, and the mean values were compared by one-way analysis of variance and Mann-Whitney U-test. The distance between root apex and maxillary sinus floor was the greatest in maxillary first premolars and shortest in the mesio-buccal roots of maxillary second molars. The distances from root apex to the buccal and palatal cortical bones were significantly greater in male patients than those in female patients (P < 0·05). The palatal roots of maxillary first molars exhibited the highest incidence as well as the greatest mean length (1·96 mm) of protrusion into the maxillary sinus. The distance from root apex to the sinus floor was found to increase with age, except in case of maxillary second premolars. Understanding the relationship of maxillary posterior teeth with the sinus floor and buccal cortex could provide clinicians valuable information to help reduce iatrogenic damage.

'Screw-in' tendency of rotary nickel-titanium files due to design geometry.

AIM: To examine the effect of several standard geometric characteristics of rotary instruments on the 'screw-in' forces and stresses generated on root dentine using 3D finite element analysis (FEA). METHODOLOGY: Four cross-sectional designs (triangular, slender-rectangular, rectangular and square) were evaluated. The area of the triangular cross-section and of the slender-rectangular model were the same. Another rectangular model had the same centre-core diameter as the triangular one. Each design was twisted into a file model with 5, 10 or 15 threads over its 16-mm-long working section. Three curved root canals were simulated as rigid surface models: θ = 15 degrees/R = 36 mm radius; θ = 30/R = 18; and θ = 45/R = 12. A commercial FEA package was used to simulate the file rotating in the canal to determine the 'screw-in' force and reaction torque on the instrument. RESULTS: Instruments of a square cross-section had the highest 'screw-in' force and reaction torsional stresses followed by the rectangle, the triangle design and the slender-rectangle design, respectively. The file with closer pitch generated lower stresses, compared with that with longer pitch. The greater the root canal curvature, the higher the 'screw-in' force and reaction torque generated. CONCLUSION: This study demonstrated that the 'screw-in' tendency depends on both the instrument geometry and canal curvature. Clinicians should be aware that certain instrument designs are prone to develop high 'screw-in' forces, requiring the operator to maintain control of the handpiece or to use a brushing action to prevent instruments being pulled into the canal.

Whole Genome Association Study to Detect Single Nucleotide Polymorphisms for Behavior in Sapsaree Dog (Canis familiaris).

The purpose of this study was to characterize genetic architecture of behavior patterns in Sapsaree dogs. The breed population (n = 8,256) has been constructed since 1990 over 12 generations and managed at the Sapsaree Breeding Research Institute, Gyeongsan, Korea. Seven behavioral traits were investigated for 882 individuals. The traits were classified as a quantitative or a categorical group, and heritabilities (h(2)) and variance components were estimated under the Animal model using ASREML 2.0 software program. In general, the h(2) estimates of the traits ranged between 0.00 and 0.16. Strong genetic (r G ) and phenotypic (r P ) correlations were observed between nerve stability, affability and adaptability, i.e. 0.9 to 0.94 and 0.46 to 0.68, respectively. To detect significant single nucleotide polymorphism (SNP) for the behavioral traits, a total of 134 and 60 samples were genotyped using the Illumina 22K CanineSNP20 and 170K CanineHD bead chips, respectively. Two datasets comprising 60 (Sap60) and 183 (Sap183) samples were analyzed, respectively, of which the latter was based on the SNPs that were embedded on both the 22K and 170K chips. To perform genome-wide association analysis, each SNP was considered with the residuals of each phenotype that were adjusted for sex and year of birth as fixed effects. A least squares based single marker regression analysis was followed by a stepwise regression procedure for the significant SNPs (p<0.01), to determine a best set of SNPs for each trait. A total of 41 SNPs were detected with the Sap183 samples for the behavior traits. The significant SNPs need to be verified using other samples, so as to be utilized to improve behavior traits via marker-assisted selection in the Sapsaree population.

Combined mesophilic anaerobic and thermophilic aerobic digestion process for high-strength food wastewater to increase removal efficiency and reduce sludge discharge.

In this study, a process that combines the mesophilic anaerobic digestion (MAD) process with thermophilic aerobic digestion (TAD) for high-strength food wastewater (FWW) treatment was developed to examine the removal of organic matter and methane production. All effluent discharged from the MAD process was separated into solid and liquid portions. The liquid part was discarded and the sludge part was passed to the TAD process for further degradation. Then, the digested sludge from the TAD process was recycled back to the MAD unit to achieve low sludge discharge from the combined process. The reactor combination was operated in two phases: during Phase I, 40 d of total hydraulic retention time (HRT) was applied; during Phase II, 20 d was applied. HRT of the TAD process was fixed at 5 d. For a comparison, a control process (single-stage MAD) was operated with the same HRTs of the combined process. Our results indicated that the combined process showed over 90% total solids, volatile solids and chemical oxygen demand removal efficiencies. In addition, the combined process showed a significantly higher methane production rate than that of the control process. Consequently, the experimental data demonstrated that the combined MAD-TAD process was successfully employed for high-strength FWW treatment with highly efficient organic matter reduction and methane production.

Expression analysis of human ß-secretase in transgenic tomato fruits.

An emerging strategy in biomanufacturing involves using transgenic plants to express recombinant pharmaceutical and industrial proteins in large quantities. ß-Site APP cleaving enzyme 1 (ß-secretase 1, BACE1) is an enzyme involved in the abnormal production of Aß42, the major component of senile plaques in Alzheimer's disease (AD). Thus, BACE1 represents a key target protein in the development of new potential drugs to treat Alzheimer's disease. We aimed to develop a tomato-derived recombinant BACE1 (rBACE1) protein to serve as a vaccine antigen that would promote an immune response. We utilized a plant expression cassette, pE8BACE, to optimize BACE1 expression in tomato fruits. Polyemerase chain reaction and Southern blot analyses verified integration of the BACE1 gene into the plant genome. Northern and Western blot analyses demonstrated successful mRNA and protein expression of rBACE1, respectively; the Sensizyme assay kit estimated the expression level of rBACE1 protein at 136 ± 7 ng mg⁻¹ total soluble protein. The tomato-derived rBACE1 retains its activity for a long storage period at cool or room temperature, and is highly resistant to degradation in conditions such as low acidity. Tomato-derived rBACE1 was severely degraded by heat or boiling. The proteolytic activity of tomato-derived rBACE1, confirmed by fluorescence resonance transfer assay, was similar to that of a commercial sample of Escherichia coli-derived BACE1.

Estimation of Effective Population Size in the Sapsaree: A Korean Native Dog (Canis familiaris).

Effective population size (Ne) is an important measure to understand population structure and genetic variability in animal species. The objective of this study was to estimate Ne in Sapsaree dogs using the information of rate of inbreeding and genomic data that were obtained from pedigree and the Illumina CanineSNP20 (20K) and CanineHD (170K) beadchips, respectively. Three SNP panels, i.e. Sap134 (20K), Sap60 (170K), and Sap183 (the combined panel from the 20K and 170K), were used to genotype 134, 60, and 183 animal samples, respectively. The Ne estimates based on inbreeding rate ranged from 16 to 51 about five to 13 generations ago. With the use of SNP genotypes, two methods were applied for Ne estimation, i.e. pair-wise r (2) values using a simple expectation of distance and r (2) values under a non-linear regression with respective distances assuming a finite population size. The average pair-wise Ne estimates across generations using the pairs of SNPs that were located within 5 Mb in the Sap134, Sap60, and Sap183 panels, were 1,486, 1,025 and 1,293, respectively. Under the non-linear regression method, the average Ne estimates were 1,601, 528, and 1,129 for the respective panels. Also, the point estimates of past Ne at 5, 20, and 50 generations ago ranged between 64 to 75, 245 to 286, and 573 to 646, respectively, indicating a significant Ne reduction in the last several generations. These results suggest a strong necessity for minimizing inbreeding through the application of genomic selection or other breeding strategies to increase Ne, so as to maintain genetic variation and to avoid future bottlenecks in the Sapsaree population.

Early line-probe assay using DNA specimens in patients with pulmonary TB.

BACKGROUND: We investigated the feasibility of early line-probe assay (LPA) using remnant DNA of Mycobacterium tuberculosis from polymerase chain reaction (PCR) test.METHODS: M. tuberculosis DNA specimens with cycle threshold (Ct) values reported and collected from patients with known results for both LPA with culture isolates and phenotype drug susceptibility testing (pDST) were selected. The diagnostic performance of DNA-based LPA according to the Ct value was investigated.RESULTS: A total of 143 respiratory specimens were included. For isoniazid resistance, the accuracy in subgroups with Ct value <25, 25-29 and ≥29 was respectively 96.8%, 65.7% and 13.3%. For rifampicin resistance, accuracy in subgroups with Ct values <29 and ≥29 was respectively 87.8% and 13.3%. When compared to the pDST results, sensitivity, specificity, positive predictive value and negative predictive value in specimens with Ct values <25 was respectively 1.00 (95% CI 0.69-1.00), 0.95 (95% CI 0.76-1.00), 0.91 (95% CI 0.59-1.00) and 1.00 (95% CI 0.83-1.00) for isoniazid resistance. For rifampicin resistance, corresponding values in subgroups with Ct values <29 were respectively 0.89 (95% CI 0.52-1.00), 0.98 (95% CI 0.91-1.00), 0.80 (95% CI 0.50-0.94) and 0.99 (95% CI 0.92-1.00).CONCLUSIONS: DNA-based early LPA with remnant DNA from respiratory samples was feasible and accurate when the Ct values were low.

Root Canal Configuration of Burmese (Myanmar) Maxillary First Molar: A Micro-Computed Tomography Study.

AIM: To investigate the root canal anatomy of Burmese (Myanmar) permanent maxillary first molar (BMFM) with micro-computed tomography. Methodology. One hundred and one extracted BMFMs were scanned by a SkyScan 1272 scanner (Bruker microCT, Belgium) and reconstructed with NRecon software (Bruker microCT). CTAn software (Bruker microCT) was used to create 3D models of root and internal canal anatomy, while CTVol software (Bruker microCT) was used to visualize 3D models. In each root, Vertucci's canal types, incidence and location of the lateral canal, incidence, location, and type of isthmus, and number and position of foramina were examined. RESULTS: In 101 specimens, 83 (82.18%) mesiobuccal roots had multiple canals. The most common canal type is type IV (45.5%), followed by type II (17.8%) and I (17.8%) canals. Type III, V, VI, VII, and VIII canals are less than 10% in total. Seven additional canal types were seen for 10% in total. Fourteen (13.86%) distobuccal roots had multiple canals, and the predominant canal type is type I (86.1%), followed by type II (5.9%) and V (4%) canals. Three additional canal types were observed for 4% in total. All palatal roots possessed the simplest type I canal. Apical ramification occurred in 69 mesiobuccal roots (68.3%), 36 distobuccal roots (35.6%), and 37 palatal roots (36.6%). A total of 240 lateral canals were observed in 101 specimens. Each specimen had 2.38 ± 2.22 lateral canals on average. The highest incidence, 136 (56.67%) lateral canals, occurred in the mesiobuccal root, followed by 57 (23.75%) and 47 (19.58%) lateral canals from the distobuccal root and the palatal root, respectively. Each specimen had 6.17 ± 2.42 foramina. Mesiobuccal root had the highest incidence of apical foramina compared to other roots. Seventy-two mesiobuccal roots (71.29%) had isthmus, while only 7 distobuccal roots (6.93%) had isthmus somewhere along the root. CONCLUSIONS: The root canal anatomy of BMFM was quite complex, especially in the mesiobuccal root. The predominant canal type was Vertucci type IV in the mesiobuccal root and type I in the distobuccal and palatal roots. In addition, this micro-computed tomography study disclosed complemented canal types and a higher prevalence of lateral canal than the previous studies.

Revisiting straight-line repair in unilateral complete cleft lip: a comparison with rotation-advancement repair.

Rotation-advancement repair (RAR) has been the most widely used technique for unilateral cleft lip repair. We recently used a straight-line repair with medial orbicularis muscle lengthening (SLR-ml) technique, based on the hypothesis that it could minimize the postoperative scar appearance without causing s short-lip deformity when muscle reorientation is performed correctly. A retrospective cohort study was conducted on unilateral complete cleft lip patients who underwent cheiloplasty between 2009 and 2017. Two cheiloplasty techniques were compared: RAR and SLR-ml. Outcomes were evaluated by assessing follow-up photographs using three methods: (1) glance impression on a five-point scale, (2) Manchester Scar Scale, and (3) indirect anthropometry. Seventy-one patients were analysed: 41 in the RAR group (28 male, 13 female) and 30 in the SLR-ml group (15 male, 15 female). The glance impression (P=0.506) and Manchester Scar Scale (P=0.347) scores did not differ between the groups. According to the symmetry ratio (cleft side value/non-cleft side value), vertical lip height (P=0.804), horizontal lip length (P=0.881), and Cupid's bow width (P=0.122) did not differ significantly between the groups. The preoperative lip height discrepancy was not correlated with the postoperative vertical lip height. The SLR-ml method can be regarded as a successful tool for symmetric repair of unilateral cleft lip.

Effects of gonadotropins on in vitro maturation and of electrical stimulation on parthenogenesis of canine oocytes.

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 mus with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 mus without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.

Impacts of pre-rigor salting with KCl on technological properties of ground chicken breast.

The objective of this study was to determine the impact of pre-rigor salting using KCl on the technological properties of ground chicken breast. Chicken breast muscle (M. pectoralis major and minor) was hot-debonded and salted with 2% NaCl (w/w), 1% NaCl+1% KCl mixture, or 2% KCl, respectively, within 30 min after slaughter. Post-rigor salting treatment was prepared with 2% NaCl at 24 h postmortem. All pre-rigor salting treatments showed higher ultimate pH, protein solubility, and final yield than post-rigor salting treatment (P < 0.05). However, the positive effects of pre-rigor salting on chicken breast differed by salt type. Pre-rigor salting with KCl resulted in higher ultimate pH and R-values of chicken breast than pre-rigor salting with NaCl (P < 0.05). Despite the high ultimate pH, pre-rigor salting with KCl resulted in lower protein solubility, final yield, and hardness of chicken breast than pre-rigor salting with NaCl (P < 0.05). These results indicate that pre-rigor salting with KCl could contribute to the maintenance of relatively excellent technological properties of pre-rigor chicken breasts compared to post-rigor salted chicken breast. However, this current study also suggests that the impact of KCl on technological properties in pre-rigor chicken breast, such as water-holding capacity, protein solubility, and texture, could be less effective than pre-rigor salting with NaCl at an identical percentage concentration.

Effect of heat-killed Enterococcus faecalis EF-2001 on ethanol-induced acute gastric injury in mice: Protective effect of EF-2001 on acute gastric ulcer.

Enterococcus faecalis is a facultative anaerobic gram-positive commensal bacterium common in the gastrointestinal tract of animals and humans. This study aimed to investigate the protective effects of heat-killed E. faecalis EF-2001 (EF-2001) on acute gastric ulcer using a murine model of ethanol (EtOH)-induced acute gastric injury. EF-2001 (20, 40, and 80 mg/kg/day) was administered by oral gavage for 5 days before EtOH treatment (10 mL/kg body weight). EF-2001 effectively attenuated EtOH-induced gastric mucosal injury with reduced gastric mucosal ulcer and histological damage score. Pretreatment of EF-2001 markedly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38MAPK). In addition, EF-2001 significantly inhibited phosphorylation of nuclear factor kappa B (NF-κB) and subsequently suppressed the upregulation of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 in gastric tissues. Taken together, these results suggest that EF-2001 exerts a gastroprotective effect against acute gastric injury, and the underlying mechanism might be associated with the suppression of MAPKs and NF-κB signaling and consequent reduction of pro-inflammatory mediators or cytokines.

Parthenogenetic induction of canine oocytes by electrical stimulation and Ca-EDTA.

In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 mus with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed.

Abscisic acid-mediated phytochrome B signaling promotes primary root growth in Arabidopsis.

Plant photomorphogenic responses have been studied mostly using the shoots, the core part of plant architecture that perceives light for photosynthesis and influences the overall processes of growth and development. While the roots are also known to respond to aboveground light through multiple routes of light signal transduction, root photomorphogenesis has been less highlighted until recently. A long-standing, critical question was how the underground roots are capable of sensing aerial light and how the root-sensed light signals trigger root photomorphogenesis. When the roots are directly exposed to light, reactive oxygen species (ROS) are rapidly produced to promote primary root elongation, which helps the roots to escape from the abnormal growth conditions. However, severe or long-term exposure of the roots to light causes ROS burst, which impose oxidative damages, leading to a reduction of root growth. We have recently found that phytochrome B (phyB) promotes abscisic acid (ABA) biosynthesis in the shoots and the shoot-derived ABA signals mediate ROS detoxification in the roots, lessening the detrimental effects of light on root growth. On the basis of these observations we propose that the phyB-mediated ABA signaling contributes to the shoot-root synchronization that is essential for optimal growth and performance in plants.

Nitrification and denitrification in partially aerated biological aerated filter (BAF) with dual size sand media.

A 104-mm (4-inch) diameter pilot-scale biological aerated filter (BAF) with a media depth of 2.5 m (8.3 feet) was operated with an anaerobic, anoxic and oxic zone at a temperature of 23 degrees C. The medium for the anaerobic and anoxic zones was 10 mm diameter sand while the medium for the oxic zone was 5 mm diameter sand. The influent sCOD and total nitrogen concentrations in the feedwater were approximately 250 mg/L and 35 mg N/L, respectively. sCOD removal at optimum hydraulic retention time (HRT) of 3 h with recirculation rates of 100, 200 and 300% in the column was above 96%. Nitrification was found to be more than 96% for 3 h HRT at 200 and 300% recirculation. Total nitrogen removal was consistent at more than 80% for 4 and 6 h HRT at 300% recirculation. For 3 h HRT and 300% recirculation, total nitrogen removal was approximately 79%. The ammonia loading rates for maximum ammonia removed were 0.15 and 0.19 kg NH3-N/m3-day for 100 and 200% recirculation, respectively. The experimental results demonstrated that the BAF can be operated at an HRT of 3h with 200-300% recirculation rates with more than 96% removal of sCOD and ammonia and at least 75% removal of total nitrogen.

Role of the hydrophobic effect in stability of site-specific protein-DNA complexes.

The site-specific binding interaction of lac repressor with a symmetric operator sequence and of EcoRI endonuclease with its specific recognition site both exhibit a characteristic dependence of equilibrium binding constant (Kobs) on temperature, in which Kobs attains a relative maximum in the physiologically relevant temperature range. This behavior, which appears to be quite general for site-specific protein-DNA interactions, is indicative of a large negative standard heat capacity change (delta C0P,obs) in the association process. By analogy with model compound transfer studies and protein folding data, we propose that this delta C0P,obs results primarily from the removal of non-polar surface from water in the association process. From delta C0P,obs we obtain semiquantitative information regarding the change in water-exposed non-polar surface area (delta Anp) and the corresponding hydrophobic driving force for association (delta G0hyd): delta G0hyd approximately equal to 8(+/- 1) x 10(1) delta C0P,obs approximately equal to -22(+/- 5) delta Anp. We propose that removal of non-polar surface from water (the hydrophobic effect) and release of cations (the polyelectrolyte effect) drive the thermodynamically unfavorable process (e.g. conformational distortions) necessary to achieve mutually complementary recognition surfaces (at a steric and functional-group level) in the specific complex.

Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle.

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.