The average age of the patients across the studies was 82, with most (71%) being female. The population had a high ABT 263 burden of comorbidity, with 32% experiencing falls, 39% dementia, 25% coronary heart disease, 28% cerebrovascular disease, and 23% diabetes mellitus. The prevalence of hypertension in care home residents as reported by these studies varied between a minimum of 16%24 and a maximum of 71%.17, 18 and 22 The mean prevalence of hypertension across the

studies was 35% (SD 18.4%). The prevalence increased over time, when later studies and earlier studies were compared, the lowest estimate being 16% in 199124 and the highest being 71% in 201022 (correlation coefficient: 0.682, Roscovitine P = .004). Of the 9 studies11, 12, 13, 14, 16, 17, 18, 19 and 22 that reported details of treatment, between 70% and 100% of their participants were on at least one antihypertensive agent. Combined across all the studies, a mean of 72% were on at least one antihypertensive agent. Overall, diuretics (27%, range 24%–66%), calcium channel blockers (26%, range 18%–30%), and angiotensin-converting enzyme inhibitors/angiotensin

receptor blockers (ACEi/ARBs) (24.6%, range 22%–65%) were most commonly used, whereas β-blockers were less commonly used (10.8%, range 8%–75%). A higher proportion of the hypertensive care home population took ACEi/ARBs (correlation coefficient: 0.875, R2 = 0.736, P = .001) and β-blockers (correlation

coefficient: 0.654, R2 = 0.427, P = .04) in later studies than in earlier studies, whereas the use of calcium channel blockers and diuretics remained static over time. There was a significant increase in the number of antihypertensive classes prescribed, when older studies were compared with more recent studies, from an average of 1.1 in 1994 to 2.0 in 2007 (correlation coefficient: 0.770, P = .025), with the median increasing P-type ATPase from 1 in 1994 to 2 in 2010. When results from these studies were combined, 70% of those with hypertension had blood pressure readings within the target range. This compared to figures of 49% on treatment in the US population (1994) with 22% reaching target blood pressures26 and 63% on treatment with 27% reaching target levels as recorded in the National Health and Nutrition Examination Survey (NHANES) database 1999–2000.27 Blood pressure control was no better in recent studies compared with older studies, and there is a trend toward poorer control over time (correlation coefficient: –0.671, R2 = 0.450, P = .099). The review demonstrated that hypertension is common in care home residents and is often treated. The prevalence of hypertension is higher in later studies than in earlier studies. The number of antihypertensive classes used per patient increased over time and the classes of antihypertensives used differed in more recent studies compared with older studies.

The preliminary finding suggest that physiologically harmful pH changes in rodent lungs after a few cryogenics-free hp gas deliveries are not likely, even with the high Rb density at 83Kr SEOP conditions and in the absence of gas filters. Although filter usage may still be prudent for selleck compound further reducing any potentially

remaining Rb contamination, a study detailing the exact quantity of the Rb carried through the gas extraction process and the effects of filtering techniques upon the spin polarization is beyond the scope of this work. Extraction scheme 2 was modified to generate hp gas mixtures with a precisely selected O2 concentration. After transfer of the hp gas into the volume Vextmax of the extraction unit, O2 was added and resulted in a carefully regulated pressure increase buy ABT-888 until the desired O2 concentration was

reached. The total pressure in the large volume Vextmax = 790 ml was typically between 10–20 kPa and the mixing of the gasses was sufficient within 5 s after addition of O2. The method was tested by measuring the 129Xe longitudinal relaxation rates caused by paramagnetic O2 as a function of O2 density (or corresponding oxygen concentration; shown in Fig. 7). The O2 density dependent relaxation data shown in Fig. 7a (filled triangles) demonstrated the accuracy in the preparation of the gas mixture. The data was obtained using a series of small flip angle pulses at physiologically relevant, (i.e. ambient) pressure. The resulting slope of the oxygen density dependent 129Xe relaxation rate equation(2) 1T1ρO2129Xe290K,9.4T=0.360±0.007s-1amagat-1at 9.4 T field strength and 290 K was in good agreement with that obtained by Jameson et al.

with thermally polarized 129Xe at high xenon and oxygen densities [31]: equation(3) 1T1ρO2129Xe9.4T=0.343s-1amagat1·(T/300K)-0.03where T   is the temperature of the gas mixture in Kelvin. An amagat is defined in this work as the density of an ideal gas at standard pressure and temperature of 101.325 kPa and 273.15 K and therefore 1amagat=2.6868×1025m-3. At the conditions used in this work, N2, O2, Kr, and Xe are considered to follow ideal gas laws. According to Eq. (2), a relaxation time of T1 = 14.2 s was observed for a 21% O2, 79% hp 129Xe–N2 mixture contained not in an NMR test tube at 9.4 T and ambient pressure. However, the experimental setup used in this work was also applied to relaxation measurements in lungs as shown in Fig. 7c after SEOP, hp gas extraction, mixing with a quantified amount of O2, compression, transfer into a storage container, and inhalation by the excised lungs. The average longitudinal relaxation rate for two excised lungs was found to have the following dependence: equation(4) 1T1ρO2129Xe290K,9.4T=0.361±0.020s-1amagat-1 Eq. (4) describes the oxygen dependent term of the 129Xe T  1 relaxation, however the average longitudinal relaxation rate measured in the absence of oxygen (i.e.

This prevented a mediastinal seroma from forming and allowed fluid to drain

into the pleural space. It also enhanced lung re-expansion by collapsing the mediastinal space. All patients had a fundoplication tailored to the patient’s esophageal manometry; it was either a complete 360-degree Nissen or a Toupet partial fundoplication. Crural tension was evaluated by visual assessment and haptic feedback. Palbociclib research buy If attempts to bring the crural pillars together with graspers were difficult or impossible, a relaxing incision was performed in the right, left, or both hemidiaphragms, as previously described.4 and 5 When less than 3 cm of intra-abdominal esophagus was present after mediastinal mobilization a wedge-fundectomy, Collis gastroplasty

was performed as previously described.6 and 7 In all patients, the crura were closed primarily using pledgeted 0-Ethibond (Ethicon) horizontal mattress sutures. The pledgets were cut from the sides of the 7 × 10 cm unhydrated AlloMax graft before its use for crural reinforcement. After crural closure, the AlloMax patch was cut into a heart-shaped pattern and placed posterior to the esophagus (Fig. 1). The graft was secured with absorbable LY2109761 cost tacks (AbsorbaTack, Covidien) or more commonly, 2-0 silk sutures and Tisseel glue (Tisseel Fibrin Sealant, Baxter International Inc). Comparisons between groups were performed using the chi-square test. A p value see more less than 0.05 was considered statistically significant. There were 82 patients (26 men and 56 women), with a median age of 63 years, who had hiatal hernia repair with an AlloMax graft reinforcement of the primary crural closure. The majority of operations (85%) were primary repairs done laparoscopically (Table 1). There was no difference in the type of fundoplication performed in patients with a PEH vs those with a sliding hiatal hernia, but patients undergoing repair of a PEH were significantly more likely

to have a Collis gastroplasty or crural relaxing incision. Crural relaxing incisions (8 right sided, 1 left sided, 1 bilateral) were necessary to achieve tension-free primary crural closure in 21% of patients with a PEH. There were 5 patients who had both a Collis gastroplasty and a relaxing incision performed. Of these, 4 were patients undergoing primary repair and 1 was a reoperation. There were 6 re-do operations for recurrent hiatal hernia and failed fundoplication. Adjunct techniques in these patients included Collis gastroplasty in 3 patients and a relaxing incision in 1 patient. Perioperative morbidity was uncommon and typically minor (Table 2). One patient underwent laparoscopic re-exploration for a falling hematocrit. A blood clot along the greater curvature of the stomach was evacuated but no source of bleeding was identified, and the patient subsequently recovered without incident. One patient had a stent placed for a leak from the Collis staple line.

The authors are grateful to , New Delhi, India, for financial assistance (SRF) to Naresh Kumar. “
“For the past 30 years, the number of promising feedstocks

for biofuels (ethanol and biodiesel) production in the US has increased E7080 order considerably, and so have prospects for the biofuels technologies of the future. With the strong support of the US Government for renewable energies, second generation biofuels have become one of the major prospective investments of the biofuels industry sector as well as biotechnology R&D. First generation biofuels from edible crops (e.g., corn, soybean, canola) (also called conventional biofuels) have been criticized for their competing with food and feed production, especially

in the face of unexpected weather events and climate change [1]. The currently investigated and produced second generation biofuels (belonging to the group of advanced biofuels) are not competing with food/feed production in a direct way. They comprise: ethanol from cellulosic plant material, e.g., switchgrass, miscanthus, poplar, and biodiesel from oil plants, e.g., jatropha, oil palm as well as biofuel from algae. According to the Renewable Fuel Standard (RFS) that has mandated biofuels production in the US since the establishment of the Energy Policy Act of 2005, 36 billion gallons (136 billion l) of biofuels are supposed to be supplied to the market by 2022. Advanced biofuels need to constitute 58.3% of the total mandate. In 2010, the RFS was extended by RFS2, setting new standards for conventional and advanced biofuels in terms of production Tacrolimus mw volumes and life cycle greenhouse gas (GHG) emissions. Thus, for instance, cellulosic ethanol is supposed to be supplied at the volume of 16 million gallons (60.5 million l) by 2022 and to guarantee 60% CO2 savings compared to fossil fuels [2]. Due to a mismatch between the mandate requirements and the actual production of cellulosic ethanol, the mandate has been adjusted and downsized by the L-NAME HCl Environmental Protection Agency (EPA) via waivers in all previous

years. Despite that, both policy makers and scientists agree that second generation biofuels represent a prospective solution of the future and can be more viable in the long-term than conventional biofuels. One of the major problems that did not allow for the advanced biofuels technology (especially cellulosic ethanol) to develop on a large commercial scale yet is the technological impediment of breaking down plant biomass (lignin in the plant walls) and releasing carbohydrate polymers (cellulose and hemicellulose) that can be converted into fermentable sugars and further refined into fuels. In addition, new highly efficient feedstocks are being unveiled as a sustainable biofuel source that could potentially outperform the currently applied second generation biofuels feedstocks.

The z-spectrum generated using the AP approximation matched well the spectrum produced by the discretization method, except at the frequency

offsets near the water center frequency (0 ppm) and chemical shift of amine protons (1.9 ppm), indicated by the green 1 circles. Consequently, only the AP continuous AG-014699 cost approximation was used to perform the continuous model fitting for the phantom data. Fig. 2 shows the values of N required for different pulsed parameters (FA, Tpd and DC) to achieve a normalized RMS error that was less than the threshold (0.1%). The smallest and largest number of segments needed within the investigated pulsed parameter ranges was 16 and 128, respectively. For the set of pulsed parameters used in the in vitro study, 32 segments per pulse were found to be sufficient. The measured z-spectra corrected using the WASSR B0 map for different creatine concentrations and pH values are shown SB431542 order in Fig. 3a and b, respectively.

Fig. 3c shows the CESTR of the phantoms after B0 correction using the WASSR map and its corresponding error bar plot is presented in Fig. 3d. When either creatine concentration or pH value increased, the dip of the amine pool and CESTR became bigger. The largest CESTR recorded was 16.7% for the 125 mM creatine phantoms with pH 6.5. R2 values calculated using N sufficient to assure accuracy obtained from the simulation for the discretized model fitting

on the phantom data are shown in Table 1. Excellent fits were found for all the measured CEST data (R2 > 99%). The fitted spectra using continuous and discretized model-based approach for 125 mM creatine phantom at pH 6 are shown in Fig. 4a. The discretization method was able to fit the measured data with small residual errors at all saturation frequencies. Similarly to the simulated data in Fig. 1, the AP continuous method also fitted with small error, except near ωw. The fitted errors using the discretization method were substantially lower than their continuous (AP) counterparts for all the phantom data, as shown in the normalized sum of square error plot in Fig. 4b. Fig. 5 shows the fitted values of water center Carnitine palmitoyltransferase II frequency shift, ωw, calculated using the discretized and continuous model-based approaches. The results matched well to each other and also to the B0 map generated using WASSR. The RMS errors and maximum difference found when the model fitted ωw were compared with the WASSR map were about 1 and 2 Hz, respectively, for both methods. Quantification of amine proton exchange rates, Clabile, using the continuous and discretized model-based approaches is shown in Fig. 6. The difference in the CV of the fitted results (CVAP – CVdiscretized) are shown in Table 2, where positive values indicate the discretized fitted results had smaller variation than the continuous ones.

The results were expressed as hazard ratios and the corresponding 95% CIs. In addition to the relative mortality between Small molecule library the 2 FITs, the absolute mortality reduction for each FIT was estimated and compared with nonparticipants with the adjustment of self-selection bias.14 The following equation was applied: RRadjustedforself-selectionbias=Screeningrate(SR)×RRparticipants/uninvited+(1-SR)×RRnon-participants/uninvited The calculation is detailed

in Supplementary Tables 2–4. Because the stage and location of screen-detected and interval cancers are of clinical significance,15 a subsidiary analysis was performed and a comparison was made between the 2 tests using the χ2 test. Cancer was staged according to the American Joint Committee on Cancer 7th staging system.16 The colon above the level of the splenic flexure (including the splenic flexure) was defined as the proximal colon. When concurrent proximal and distal cancers were present, subjects were placed into the distal colon category. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC). All P values were 2-sided and P < .05 was considered to indicate statistical significance. Between January 1, 2004 and December 31, 2009, selleck chemicals a total of 956,005 subjects underwent screening. Among them, 747,076 (78%) and

208,929 (22%) received the OC-Sensor and HM-Jack tests, respectively; their baseline data according to demographic characteristics, geography, and temperature, and characteristics of the confirmatory diagnosis are presented in Table 1. Small differences, which were statistically significant owing to the large sample size, were observed with respect to sex, follow-up time, confirmatory examination tool, colonoscopy adenoma detection rate, and colonoscopy advanced adenoma detection rate. Differences were more prominent in the geographic areas and the hospital levels where Fossariinae confirmatory diagnoses were performed. As shown in Table 2, positivity rates were similar between the 2 tests (3.8% vs 3.9%), but the confirmatory examination rate was higher for those who received

HM-Jack (80.9% vs 85.3%). As expected, positivity rates were higher for males and those of older age as compared with the total population group. These findings were unchanged regardless of adjustments for sex and age distributions (data not shown). The effect of ambient temperature on FIT positivity was also evaluated. For the temperature ranges of 10–14°C, 15–19°C, 20–24°C, and ≥25°C, the positivity rates for OC-Sensor were 5.6%, 4.4%, 3.9%, and 3.6%, respectively, and for HM-Jack were 5.5%, 3.8%, 4.7%, and 3.6%, respectively, revealing an inverse association (P < .001) between FIT positivity and ambient temperature. The OC-Sensor test detected CRC in 0.21% of patients, with a positive predictive value of 6.8%.

The animals were housed in individual stainless steel cages with free access to a standard sodium diet (Guabi Rat Chow, Paulinia, SP, Brazil), water and 0.3 M NaCl solution. The positions of the bottles containing water and 0.3 M NaCl were rotated daily to avoid place preference. Rats were maintained in a room whose temperature was controlled at 23 ± 2 °C and humidity at in a 12-h light/dark cycle with lights on 7:30 a.m. The animals were randomly divided into two groups: the control group (CN) and the periodontal disease group (PD). Under general anaesthesia (a mixture of ketamine (80 mg/kg of

body weight (b.w.), Cristália, Brazil) and xylazine (7 mg/kg of b.w., Agener, Brazil)) injected subcutaneously, a sterile silk Roscovitine cost ligature (strength 4/0) was tied in the cervical region of the mandibular first molars teeth bilaterally in the PD group using a technique that was previously described.9 The ligatures served UK-371804 cost as a retention device for oral micro-organisms. Ingestion of 0.3 M NaCl and water (ml/24 h) was measured 3 and 16 days after experimental ligature-induced periodontal disease in order to verify the systemic conditions of the animals. On the 15th day after ligature placement, control rats (without ligature) and rats with PD were anaesthetised with i.p. injection of ketamine (80 mg/kg of b.w.) combined with xylazine (7 mg/kg of b.w.)

and placed in a stereotaxic instrument (Kopf, Tujunga, CA, USA). The skull was levelled between bregma and lambda. Stainless steel guide-cannulas (12 mm × 0.6 mm outer diameter (o.d.)) were implanted bilaterally into the LPBN using the following coordinates: 9.2 mm caudal to bregma, 2.2 mm lateral to the midline and 3.8 mm below the dura mater. The tips of the cannulas were positioned 2 mm above each LPBN. The cannulas were fixed to the cranium using dental acrylic resin and jeweller screws and were filled with 30-gauge metal obturators

between tests. After the surgery, only control rats received a prophylactic dose of the antibiotic penicillin (30,000 IU). All animals were allowed to recover for 5 days before starting ingestion tests and during this period they had free access to standard sodium diet, water and 0.3 M NaCl solution. Tryptophan synthase Bilateral injections into the LPBN were made using 5-μl Hamilton syringes connected to 30-gauge injection cannulas by means of polyethylene tubing (PE-10). At the time of testing, the obturators were removed and the injection cannula (2 mm longer than the guide cannula) was carefully inserted into the guide cannula. For bilateral injections, the first injection was performed on one side, the needle was removed and repositioned on the contralateral side and then the second injection was given. Therefore, injections were given ∼1 min apart. The injection volume into the LPBN was 0.2 μl on each site. The obturators were replaced after the injections, and the rats were put back into their cages.

Eight hours after injection, severity of mucus secretion, loss of turgor, matting of spines, and tissue necrosis ranged from low to medium. There was an increase in severity of these signs after 24 h. Even at 0.25× the standard concentration, severity of

mucus secretion, loss of turgor, matting of spines, selleck and tissue necrosis ranged from medium to severe after 24 h and resulted in 80% mortality. All sea stars were dead 48 h after injection. There was 0% mortality at the TCBS standard concentration (5 g l−1) and also when this concentration was doubled. Disease signs were not exhibited except for localized swelling and tissue necrosis at the site of injection. Twelve days after exposure to A. planci injected with oxgall (8 g l−1, 4 g l−1), peptone (20 g l−1), and TCBS (44 g l−1), none of the fish, corals, and mobile invertebrates exhibited any signs of disease. No signs of bacterial disease such as cloudy eyes, fin rot, pop eyes and changes in skin color were observed in any of the fish tested. There were also no spots, bands, or discoloration observed in corals that were constantly in contact with floating A. planci particles in the water. It is important

to mention that corals anti-EGFR antibody were not attacked by A. planci and there was minimal movement of the starfish one hour after injection with oxbile. Mobile invertebrates remained active each night and there was no loss of spines observed in sea urchins and no lesions in sea stars and sea cucumbers ( Fig. 3). Bile derivatives (i.e. oxgall, bile salts) have consistently resulted in high mortality rates in previous studies (Rivera-Posada

et al., 2012) and in this study. Bile salts are added in media culture formulations to inhibit the growth of gram-positive bacteria and isolate resistant strains. Bile is a natural digestive enzyme produced by all vertebrates to aid in the digestion of lipophilic nutrients. In addition, bile is an important route of elimination of environmental toxins, carcinogens, hormones, drugs and their metabolites and may control the growth of bacteria in the small intestine (Nathanson and Boyer, 1991). Two well-known mechanisms of cell death are triggered by bile acids: necrosis at higher concentrations and apoptosis at lower concentrations (Palmeira and Rolo, 2004 and Rolo et al., 2004). Several Histone demethylase studies indicate that impairment of mitochondrial oxidative phosphorylation is an early and critical event in the mechanism of bile acid cytotoxicity. Apoptosis induction is dependent on the bile acid, its concentration, or its conjugation state. Toxic bile acid-induced apoptosis involves both extrinsic (death receptor-mediated apoptosis) and intrinsic (direct targeting to mitochondria) apoptotic pathways. Bile acids induce alterations in membrane fluidity associated with impairment of mitochondrial respiration and mitochondrial depolarization.

2 × 104 M−1 cm−1 ( Murphy and Kehrer, 1989). The total protein content of lymphocytes was measured by the method of Bradford (Bradford, 1976), using BSA as standard. All data are expressed as mean values and standard errors of at least three independent experiments. Data were analyzed by one-way ANOVA followed by the Tukey’s post hoc test. The software employed for statistical

analyses was GraphPad Prism (version4; GraphPad Software, San Diego, CA, USA). The Selleckchem HSP inhibitor functional activity of lymphocytes was assayed by their capacity to proliferate in response to a specific stimulation. Fig. 1 shows the MTT assay results after stimulation with Con A (a T lymphocytes mitogen) or LPS (a B lymphocytes mitogen)

for 48 h. FA at 0.3 mM ERK inhibitor in vitro increased both basal (without stimulation) and LPS-stimulated proliferative capacity of human lymphocytes by 38% and 30%, respectively as compared with non stimulated control group. The addition of astaxanthin to cells treated with FA caused a decrease in the proliferation of lymphocytes in basal, Con A and LPS-stimulated conditions by 43%, 26% and 30%, respectively as compared with 0.3 mM of FA mixture. Intracellular Ca2+ mobilization was significantly enhanced by the mixture of FA in human lymphocytes (about 31-fold) when compared to the control group (Fig. 2). The increase in Ca2+ levels was sustained during 20 min of kinetic monitoring. Treatment with ASTA was unable to prevent the calcium increase induced by FA. BSA (0.2%) addition was able to partially decrease calcium mobilization probably by chelating free FA. To measure intracellular superoxide anion, hydrogen peroxide and nitric oxide production, cells were acutely treated with the FA mixture with or without ASTA as indicated in the material and methods section. As shown in Fig. 3A, the treatment of human lymphocytes with the FA mixture increased the intracellular superoxide

anion levels by 135% as compared with the PMA-control group and as assessed by using Amine dehydrogenase DHE probe. The addition of ASTA to FA-treated cells promoted a reduction of 20% in superoxide production. Treatment of PMA-control cells with DPI, a NADPH-oxidase inhibitor, totally inhibited superoxide anion production, whereas sodium azide (SA) partially inhibited superoxide anion production. DPI addition in cells treated with fatty acid mixture partially decreased (20%) the superoxide anion production (Fig. 3A). A similar pattern was observed when DCFH-DA probe was used as a general ROS probe (Fig. 3B). An increase of threefold in total ROS production was observed in lymphocytes treated with the FA mixture as compared with PMA-control group. ASTA-treatment decreased the ROS production induced by FA in 20%. Addition of BSA, used as a FA chelating agent, reduced the ROS production in about 32%.

To successfully select those residues in the active site, a theoretical model of RgPAL was constructed through homology modeling using RtPAL (PDB ID: 1T6J) as the template. As shown in Fig. 2, all of the residues that were

the superimposed with RtPAL showed an RMSD of 0.224 Å ( Fig. 2A), and the Ramachandran plot suggests that 94.9%, 3.2%, and 1.9% of the residues in derived model are in acceptable region, marginal Epigenetic activity region and disallowed region, respectively ( Fig. 2B), These finding indicated that the model is reasonable and could be used in further molecular docking simulation. Using the AutoDock global–local evolutionary algorithm, we searched for those sites with the lowest free energy of binding between the ligand and the enzyme. As shown in Fig. 3, the active site cavity of RgPAL was bisected into

two regions ( Fig. 3A): one binds the amide group adjacent to the aromatic ring and the other binds the carboxyl group of the substrate. The phenyl ring of the substrate is roughly orthogonal to the plane of the MIO, and the methylidene of the MIO points to C2 of the aromatic ring ( Fig. 3A and B). In the carboxyl group binding pocket, the Arg361 residue is 3.2 Å from the carboxyl group of the substrate, and this residue might play a role in PD332991 the binding of the carboxylate moiety of the substrate through a salt bridge. The Tyr358 residue is 2.7 Å from the β-H of substrate, which is close enough to act as the β-H abstracted base ( Fig. 3C). The Glu491 residue is the closet residue to the amino group of the substrate (2.8 Å, Fig. 3C) and might accept the amino group of substrate as the enzyme base, which is consistent with the results reported by Bartsch [1]. The Tyr358, Arg361 and Glu491 are highly conserved in PAL ( Fig. 1). In the aromatic ring binding pocket, the His136 residue points to the phenyl ring of the substrate. The imidzaole group

of His136 is parallel to the phenyl ring and might generate a π–π interaction. Moreover, the imidazole of His136 and the adjacent amide group of Gln137 which points to the phenyl ring within a distance of 4.5 Å, form a hairpin motif to clamp the phenyl ring ( Fig. 3B and C). To verify the function of the hairpin, the His136, Gln137 were deleted (RgPAL-Δ136H, RgPAL-Δ137Q) and mutated to negative (RgPAL-H136E, Grape seed extract RgPAL-Q137E) and positive charges (RgPAL-H136K, RgPAL-Q137K) as well as uncharged amino acids (RgPAL-H136F, RgPAL-Q137L), respectively. The mutant and wild type RgPAL proteins appeared a single band of about 75 kDa on SDS-PAGE ( Fig. 4). The activities of RgPAL-Δ136H and RgPAL-Δ137Q were not detected (data not shown), suggesting that the residues at the two sites were essential for catalysis. The RgPAL-H136K, RgPAL-Q137K and RgPAL-H136E lost the enzymatic activity (data not shown), and the RgPAL-H136F, RgPAL-Q137L sharply decreased the activity ( Fig. 5). Compared with those mutants, the activity of RgPAL-Q137E decreased slightly ( Fig. 5).