[Changes in serum osteoprotegerin/receptor activator of nuclear factor-κB ligand and related indexes of calcium and phosphorus in the early stage of severe burn patients].

Objective: To investigate the early changes in serum osteoprotegerin/receptor activator of nuclear factor-κB ligand (RANKL) and related indexes of calcium and phosphorus in severe burn patients. Methods: Thirty severe burn patients who met the inclusion criteria and were admitted to Tongren Hospital of Wuhan University & Wuhan Third Hospital within 8 hours post injury from June 2017 to December 2018 were recruited into severe burn group (24 males and 6 females, aged (38±13) years). Ten healthy volunteers with normal physical examination results in the Physical Examination Center of the same hospital in the same period of time were recruited into healthy control group (7 males and 3 females, aged (37±8) years). A prospective controlled study was conducted. The fasting venous blood of 5 mL was taken from each patient in severe burn group on post injury day (PID) 1, 7, 14, 21, and 28, respectively, and the fasting venous blood of 5 mL was taken from each volunteer in healthy control group. The serum osteoprotegerin, RANKL, 25 hydroxyvitamin D, and parathyroid hormone (PTH) levels were determined by enzyme-linked immunosorbent assay, and the RANKL/osteoprotegerin ratio was calculated. Serum albumin, serum calcium, and serum phosphorus levels were determined by bromocresol green method, methylthymol blue method, and phosphomolybdic acid method, respectively. Data were statistically analyzed with Fisher's exact probability test, analysis of variance for repeated measurement, Mann-Whitney U test, independent sample t test, and Bonferroni correction. Results: (1) The serum osteoprotegerin levels of patients in severe burn group on PID 1, 7, 14, 21, and 28 were 155.11 (102.91, 187.02), 170.07 (84.60, 196.86), 174.95 (59.09, 208.35), 190.01 (47.08, 214.52), and 188.85 (58.73, 223.13) pg/mL, respectively, which were significantly higher than 33.34 (28.59, 45.68) pg/mL of volunteers in healthy control group, Z=-3.436, -4.311, -3.248, -2.811, -4.217, P<0.01. The serum levels of RANKL of patients in severe burn group on PID 1, 7, 14, 21, and 28 were (1 869±791), (1 746±857), (1 781±713), (2 015±825), and (2 272±583) pg/mL, respectively, significantly higher than (49±16) pg/mL of volunteers in healthy control group, t=12.600, 10.844, 13.294, 13.041, 20.880, P<0.01. The ratios of RANKL/osteoprotegerin of patients in severe burn group on PID 1, 7, 14, 21, and 28 were 12.23 (8.10, 24.73), 11.40 (8.25, 16.96), 11.15 (6.91, 38.32), 12.98 (9.22, 49.68), and 13.91 (10.29, 40.68), respectively, which were significantly higher than 1.17 (0.91, 1.74) of volunteers in healthy control group, Z=-4.560, -4.529, -4.529, -4.560, -4.623, P<0.01. (2) The serum level of 25 hydroxyvitamin D of patients in severe burn group on PID 1 was significantly lower than that of volunteers in healthy control group (Z=-2.749, P<0.01). Compared with those of volunteers in healthy control group, the serum levels of albumin of patients in severe burn group on PID 1, 7, 14, 21, and 28 were significantly lower (t=-4.374, -7.689, -8.257, -7.651, -6.259, P<0.01), the serum levels of PTH were significantly elevated (Z=-4.685, -4.685, -4.685, -4.654, -4.685, P<0.01), and the serum levels of phosphorus were not changed significantly. The serum levels of calcium of patients in severe burn group on PID 1, 7, 14, and 21 were significantly lower than the level of volunteers in healthy control group (Z=-2.375, -3.455, -2.442, -2.016, P<0.05 or P<0.01). Conclusions: The serum osteoprotegerin, RANKL, RANKL/osteoprotegerin ratio, and PTH are increased, and the serum 25 hydroxyvitamin D, albumin, and calcium are decreased in the early stage of severe burn patients, which may be the mechanism leading to bone loss in patients.

Leucine promotes the growth of squabs by increasing crop milk protein synthesis through the TOR signaling pathway in the domestic pigeon (Columba livia).

Leucine (Leu) plays a critical regulatory role in protein synthesis, however, the effects and molecular mechanisms of Leu on crop milk protein in the domestic pigeons (Columba livia) are still unknown. Therefore, the study aimed to investigate the effects of dietary Leu supplementation on crop milk protein synthesis and the growth performance of squabs and the possible underlying mechanism. A total of 240 pairs of breeding pigeons (1102.3 ± 9.5 g/pair) were randomly assigned to 1 of 5 treatments, including a positive control (PC) diet that had adequate crude protein (crude protein, CP = 18%; Leu = 1.30%), a negative control (NC) diet that was low in CP (CP = 16%, Leu = 1.30%), and NC diets supplemented with Leu at 0.15%, 0.45%, or 1.05%. Compared with the NC diet, 0.15 to 0.45% Leu supplementation decreased BW loss and increased relative crop weight, crop thickness, and protein levels in the crop tissue and milk of breeding pigeons. However, dietary supplementation with 1.05% Leu inhibited ADFI in breeding pigeons. Dietary supplementation with 0.15 to 0.45% Leu decreased the mortality rate and increased the BW, eviscerated yield, and breast muscle yield of young squabs. The protein expression levels of the target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1), ribosomal protein S6 kinase (S6), eukaryotic initiation factor 4E binding protein 1 (4EBP1), and eukaryotic translation initiation factor 4E (eIF4E) were upregulated in the crop tissue of breeding pigeons in PC, 0.15% and 0.45% Leu-supplemented groups. Collectively, these results indicated that 0.15 to 0.45% Leu supplementation could decrease BW loss, increase milk protein synthesis in the crop of breeding pigeons, and enhance the survival rate and growth performance of young squabs through the TOR signaling pathway.

[Current status of the treatment of drug-induced liver injury].

In recent years, with the amazing increase in the number of prescription drugs and non-prescription drugs and the irrational use of Chinese herbal medicine and healthcare products, the incidence of drug-induced liver injury (DILI) has been increasing year by year; however, there is still a lack of effective therapeutic methods for DILI at present. Standard treatment based on timely diagnosis should be emphasized, and it is necessary to fully evaluate the severity of liver injury and primary disease, withdraw suspected drugs in time, and select appropriate drugs. Liver transplantation should be recommended to patients with severe liver failure.

Meta-analysis of traditional herbal medicine in the treatment of nonalcoholic fatty liver disease.

Traditional Chinese herbal medicine (TCM) has been found effective for nonalcoholic fatty liver disease (NAFLD) based on its unique theory system and substantial herb remedies. The aim of this meta-analysis was to evaluate the efficiency and safety of one of the TCM, Danshen in the treatment of NAFLD. Seven English and Chinese databases were searched from inceptions to December 2015. RCTs which compared Danshen with placebo in adult patients with NAFLD were included. Totally, 8 RCTs with a total of 800 patients were identified. The results showed that compared with placebo, Danshen had increased total effectiveness rate, lower level of ALT, AST, TC and TG, LDL and higher level of liver/spleen computed tomography ratio. The present meta-analysis suggests that Danshen may have positive effects on nonalcoholic fatty liver disease. Future multicenter large-sample randomized clinical trials are still needed to confirm the efficacy and safety of our study.

The screening and evaluation of herbs and identification of herbal combinations with anti-viral effects on Newcastle disease virus.

A total of 25 "heat-clearing and detoxifying" herbs used in Chinese medicine were investigated for their cytopathic effects on the growth of Newcastle Disease virus (NDV) in a chicken fibroblast cell line. The 5 herbs with the highest virus inhibitory effects were Herba agastaches, Flos chrysanthemi indici, Rhizoma anemarrhenae, Astragalus root and Baikal skullcap root and these were used in herbal formulations. Anti-NDV activities of 4 formulations were tested on the growth of NDV in the DF-1 fibroblast cell line. Formulation II, containing Baikal skullcap root, Astragalus root, Anemarrhena rhizome (1:1:2) and formulation IV containing Anemarrhena rhizome, Astragalus root and Flos chrysanthemi indici (1:1:1), which had strong anti-NDV activity in vitro, were used to determine the in vivo inhibitory effects of NDV-infection in chickens. After treatment with the two formulations serum IgY titres against NDV were improved, and morbidity was reduced in the NDV-infected chickens. The results suggest that the components in formulations II and IV acted synergistically to improve resistance to Newcastle disease and provide a basis for the developing an anti-NDV herbal medicine.

The expression and function of Frizzled-7 in human renal cell Carcinoma

Purpose: Wnt/b-catenin has emerged as an important signal pathway in renal cell carcinoma (RCC) pathogenesis. Frizzled 7 (Fzd7) is a member of Frizzled (Fzd) receptor family which binds with Wnt ligands and transduces canonical and non-canonical pathways. However, the expression of Fzd7 in human RCC is poorly investigated. Methods: 53 RCC tissues and peri-tumor tissues were collected from the patients treated with radical nephrectomy. The expression of Fzd7 was investigated by immunohistochemical staining. Three RCC cells were transfected with Fzd7shRNA and GFPshRNA to investigate the function of Fzd7 in RCC cells. Results: The immunohistochemical analysis showed that Fzd7 protein expression level was significantly increased in RCC tissues when compared with peri-tumor tissues, which suggested that Fzd7 might be involved in the formation of tumors. However, the Fzd7 expression was not correlated with clinicopathological parameters. Three RCC cell lines: 786-O, Caki-1, and OS-RC-2 also expressed Fzd7. With Fzd7 expression being interfered by shRNA, the RCC cell proliferation was mildly decreased. Wnt3a could stimulate the RCC cells proliferation, but the stimulation was decreased when Fzd7 expression was interfered. Restoring the Fzd7 expression led to the proliferation stimulation effect of Wnt3a being restored. Conclusions: This paper suggests that Fzd7 may act as one of the molecules that take part in the course of RCC formation. Fzd7 can be activated by Wnt3a to stimulate cell proliferation (AU)

No disponible

Nosocomial pneumonia caused by carbapenem-resistant Raoultella planticola: a case report and literature review.

Raoultella planticola is a rare opportunistic pathogen usually invaded immunocompromised patients and sometimes even causes fatal infections. Recently, there is growing concern about the emergence of carbapenem resistance in this species. Here, we describe one case of hospital-acquired pneumonia due to a carbapenem-resistant R. planticola (CRRP) co-producing Klebsiella pneumoniae carbapenemase and extended-spectrum ß-lactamase. A literature review was performed to indicate the microbiological and clinical features of infections caused by CRRP.

Diabetes is an inflammatory disease: evidence from traditional Chinese medicines.

Diabetes is usually associated with inflammation. Inflammation contributes to the development of diabetes. Traditional Chinese medicines (TCM) play an important role in lowering blood glucose and controlling inflammation. Many studies show that TCM with hypoglycaemic effects, for example Radix Astragali, Radix Rehmanniae, Radix Trichosanthis, Panax Ginseng, Fructus Schisandrae, Radix Ophiopogonis, Rhizoma Anemarrhenae, Radix Puerariae, Fructus Lycii, Poria, Rhizoma Coptidis, Rhizoma Dioscoreae, Rhizoma Polygonati, Radix Salviae Miltiorrhizae, Radix Glycyrrhizae, Semen Trigonellae, Momordica charantia, Allium sativum, Opuntia stricta, Aloe vera, Cortex Cinnamomi, Rhizoma Curcumae Longae, and so on, have nearly independent anti-inflammatory action. Antihyperglycaemic compounds, for example berberine, puerarin, quercetin, ferulic acid, astragaloside IV, curcumin, epigallocatechin gallate, resveratrol, tetrandrine, glycyrrhizin, emodin and baicalin, used in TCM also have anti-inflammatory effects. These studies suggest that TCM might exert hypoglycaemic effects that are partly mediated by the anti-inflammatory mechanisms. However, small amounts of TCM with potent anti-inflammatory action does not have any hypoglycaemic effect. This indirectly indicates that diabetes may be a low-grade inflammatory disease and potent regulation of inflammatory mediators may not be required. Studies of TCM add new evidences, which indicate that diabetes may be an inflammatory disease and slight or moderate inhibition of inflammation might be useful to prevent the development of diabetes. Through this review, we aim to develop more perspectives to indicate that diabetes may be an inflammatory disease and diverse TCM may share a common antidiabetic property: anti-inflammatory action. Further studies should focus on and validate inflammation-regulating targets of TCM that may be involved in inhibiting the development of diabetes.

Evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice.

OBJECTIVE: To investigate the anti-obesity effects of the pomegranate leaf extract (PLE) in a mouse model of high-fat diet induced obesity and hyperlipidemia. DESIGN: For the anti-obesity experiment, male and female ICR mice were fed with a high-fat diet to induce obesity. When the weight of the high-fat diet group was 20% higher than the normal diet group, the animals were treated with 400 or 800 mg/kg/day of PLE for 5 weeks. Body weight and daily food intake were measured regularly during the experimental period. The various adipose pads were weighed and serum total cholesterol (TC), triglyceride (TG), glucose and high-density lipoprotein cholesterol (HDL-C) were measured after 5 weeks, treatment with PLE. In the fat absorption experiment, both the normal and obese mice were given 0.5 ml lipid emulsion and PLE at a dose of 800 mg/kg at the same time. Serial serum TG levels were measured at times 1, 2, 3, 4 and 6 h after the treatment. TGs in fecal excretions were measured after the mice were orally given a lipid emulsion. Effects of PLE and its isolated compounds (ellagic acid and tannic acid) on pancreatic lipase activity were examined in vitro. RESULTS: The PLE-treated groups showed a significant decrease in body weight, energy intake and various adipose pad weight percents and serum, TC, TG, glucose levels and TC/HDL-C ratio after 5 weeks treatment. Furthermore, PLE significantly attenuated the raising of the serum TG level and inhibited the intestinal fat absorption in mice given a fat emulsion orally. PLE showed a significant difference in decreasing the appetite of obese mice fed a high-fat diet, but showed no effect in mice fed a normal diet. CONCLUSION: PLE can inhibit the development of obesity and hyperlipidemia in high-fat diet induced obese mice. The effects appear to be partly mediated by inhibiting the pancreatic lipase activity and suppressing energy intake. PLE may be a novel appetite suppressant that only affects obesity owing to a high-fat diet.

alpha(1)-adrenergic receptors mediate LH-releasing hormone secretion through phospholipases C and A(2) in immortalized hypothalamic neurons.

Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-releasing hormone (LHRH). In vivo and in vitro studies indicate that these effects are mediated primarily through alpha(1)-adrenergic receptors (alpha(1)-ARs). With the immortalized hypothalamic LHRH neurons, we have found that alpha(1)-adrenergic agents directly stimulate the secretion of LHRH in a dose-dependent manner. Ligand binding and RNA studies demonstrate that the GT1 cells contain both alpha(1A)- and alpha(1B)-ARs. Competition binding experiments show that approximately 75% of the binding is due to alpha(1B)-ARs; the remainder is made up of alpha(1A)-ARs. Receptor activation leads to stimulation of PLC. PLC beta 1 and PLC beta 3 are expressed in GT1 neurons, and these PLCs are probably responsible for the release of diacylglycerol and IP as well as the increase in intracellular calcium. The mobilization of cytoplasmic calcium is sufficient to stimulate cytosolic PLA(2) (cPLA(2)) and release arachidonic acid. A dissection of the contributions of the phospholipases to LHRH secretion suggests that cPLA(2) acts downstream of PLC and that it significantly augments the PLC-stimulated LHRH secretory response. Inasmuch as the alpha(1)-ARs are known to play a critical role in LHRH physiology, we propose that both PLC and cPLA(2) are critical in regulating and amplifying LHRH release.

[Effect of new zhengtian pill on expression of whole blood platelet membrane adhesion molecules in patients of migraine].

OBJECTIVE: To investigate the effect of New Zhengtian Pill (NZTP) on expression of whole blood platelet membrane adhesion molecules (PMAM) in patients of migraine. METHODS: Sixty-eight patients were divided into two groups, the 35 patients in the treated group treated by NZTP orally and the 33 patients in the control group treated by Fuguiqin Capsule with a therapeutic course of 30 days for both groups. Changes of PMAM GP II b/III a(CD41) and P-selectin (CD62P) were observed by flow-cytometry and compared with those in healthy persons. RESULTS: The markedly effective rate and total effective rate in the treated group was higher than those in the control group respectively (P < 0.05 and P < 0.01). The PMAM expression was also higher in patients, both at onset stage and intermittent stage, than in healthy persons (P < 0.01), NZTP treatment could reduce their increased expression significantly (P < 0.01). CONCLUSION: NZTP could reduce the PMAM expression and inhibit the activation of platelet.

[Effects of combined transdermal acupoint electric stimulation and isoflurane anesthesia on isoflurane-induced hypotension and tissue oxygen metabolism in patients undergoing craniotomy].

OBJECTIVE: To observe the effects of combined transdermal acupoint electric stimulation and isoflurane anesthesia on isoflurane-induced hypotension and tissue oxygen metabolism in patients undergoing craniotomy. METHODS: Forty-two patients with brain tumor of ASA physical status I or II and scheduled for elective surgery were randomly divided into two groups. The craniotomy was undergoing in Group A with isoflurane anesthesia, while in Group B, with combined transdermal acupoint electric stimulation (TAES) and isoflurane anesthesia. Isoflurane induced hypotension was performed in both groups by augmenting the concentration of isoflurane to lower the mean arterial pressure than before anesthesia for 30%-40% and maintain for 30-45 mins. The tissue oxygen metabolism, blood gas and arterial lactic acid level before, during and after hypotension were monitored by Swan-Ganz floating catheterization. RESULTS: As compared with before hypotension, the pulmonary arterial and mixed with venous blood oxygen saturation lowered and oxygen supply lowered, and oxygen uptake increased significantly in both groups (P < 0.05), but the extent of changes in oxygen metabolic criteria in Group B were all lesser than those in Group A (P < 0.05). CONCLUSION: Combined acupoint electric stimulation and general anesthesia could maintain the tissue oxygen supply and demand balance better in the isofluraneinduced hypotension process.

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba.

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.

Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma.

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.

Automated in-tube solid phase microextraction coupled with HPLC-ES-MS for the determination of catechins and caffeine in tea.

A polypyrrole (PPY) coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for catechins and caffeine. Compared with commercial capillaries that were currently used for in-tube solid phase microextraction (SPME), the PPY coated capillary showed better extraction efficiency for all of the compounds studied. Electrospray mass spectrometric (ES-MS) detection conditions were also investigated. After optimization of the extraction and detection conditions, a method for the sensitive and selective determination of catechins and caffeine was developed by coupling the PPY coated capillary in-tube SPME with HPLC-ES-MS. Catechins could be determined in both positive and negative ion detection modes. The detection limit (S/N = 3) for each of the studied catechins was < 0.5 ng mL-1. Caffeine could only be determined under positive ES-MS detection conditions and its detection limit was 0.01 ng mL-1. Caffeine and the five catechins in several tea samples were determined using the developed method. Small amounts of catechins were also detected in grape juice and wine samples.

[Clinical application of combined acupuncture-drug anesthesia with isoflurane-induced hypotension in cerebral operation].

OBJECTIVE: To evaluate the feasibility of combined acupuncture-isoflurane anesthesia with deliberated and controlled hypotension induced by isoflurane for the neurosurgical patients. METHODS: Forty-two patients with brain tumor scheduled for selective surgery according to the methods of anesthesia, they were further divided into isoflurane anesthesia (group I, n = 21) and combined acupuncture-isoflurane anesthesia (group II, n = 21). Anesthesia was induced with fentanyl, sodium pentothal and phi avolon intravenously administered. The concentration of isoflurane was elevated to reduce mean arterial pressure (MAP) 30%-40% during dissection and occlusion of the tumor. Compare with the hemodynamic parameters (CO, CI, SV, SI, SVR, LVSWI, RPP) which were measured with Swan-Ganz technique before, during and after isoflurane-induced hypotension. In group II, during tracheal intubation the acupoint of Quanliano (SI 18), Yuyao (EX-HN4), Fengchi (GB 20) and Hegu (LI 4) were stimulated by Han's Acupoint Nerve Stimulator. Meanwhile, the isoflurane was inhalated to maintain anesthesia. RESULTS: As compared with group I, concentration of isoflurane decreased significantly, and isoflurane average dosage per hour reduced by 31%-42% in group II. SVR, PVR, RPP were significantly decreased in the duration of hypotension (P < 0.05). The range change of CO, CI, SV, SI, LVSWI, RPP during controlled hypotension was significantly lower in combined acupuncture-isoflurane anesthesia than that in isoflurane anesthesia (P < 0.05). CONCLUSION: Combined acupuncture-isoflurane anesthesia with isoflurane induced hypotension was used as a selective way for deliberated and controlled hypotension to the neurosurgical patients.

Protective effect of gypenosides on DNA and RNA of rat neurons in cerebral ischemia-reperfusion injury.

AIM: To observe the protective effect of gypenosides (GP) on the neurons of hippocampus, cerebral cortex, corpus striatum, and dentate gyrus in cerebral ischemia-reperfusion injury of rats. METHODS: Modified 4-vessel occlusion (4-VO) method was used to establish the model of acute global ischemia. The acridine orange (AO) staining method was used to observe the DNA and RNA contents of cerebral ischemia-reperfusion injury model in the areas. RESULTS: The fluorescent intensity (reflecting DNA and RNA contents) of the DNA and RNA in the areas of cerebral ischemia-reperfusion injury was markedly abated compared with the normal control group. In the group of ig GP (100 mg/kg) it was enhanced compared with the model group and was the same as the normal control group. CONCLUSION: The injury of the DNA and RNA in the areas of ischemia-reperfusion model was decreased by GP.

[The role of ozone solution on debridement and sterilization of burn wound].

OBJECTIVE: To observe the role of ozone solution on debridement and sterilization of burn wound. METHODS: In vitro sterilizing effect on common isolated bacteria from burn wound and debridement and sterilization effects on burn wound of ozone disinfectant (ozone solution) were studied. RESULTS: All the bacteria tested were killed in vitro by ozone solution. In addition, when ozone solution was applied on burn wound, its clearance rate of bacteria was 94.5% and the clinical effective rate was 97.1%. CONCLUSION: Ozone is low in cost and high in effect which might be used as an agent for burn wound disinfection.

Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis.

Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.

Identification of residues essential for human paraoxonase (PON1) arylesterase/organophosphatase activities.

Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.