[Fifth Chinese national consensus report on the management of Helicobacter pylori infection].

The fifth national consensus conference on the management of Helicobacter pylori (H.pylori) infection organized by Chinese Medical Association, Chinese Society of Gastroenterology, H. pylori and Peptic Ulcer Study Group was held at Hangzhou, Zhejiang Province on December 15-16, 2016.More than 80 members of the study group and experts in the field of H. pylori research and gastroenterology attended the meeting.Consensus preparatory group was established to draft the related statements.The quality of evidence and strength of recommendation were evaluated by GRADE system.The Delphi method using an anonymous electronic system was adopted to develop the consensus of relevant statements.Expert advices about the established statements were firstly consulted through the emails.After two rounds of consultation, the preliminary statements of consensus were discussed and modified in the conference item by item.A total of 21 core members voted for the final version, which contained a total of 48 statements and was divided into 6 parts, including indications for H. pylori eradication, diagnosis, treatment, H. pylori and gastric cancer, H. pylori infection in special populations, H. pylori and gastrointestinal microbiota.

An instrument for measuring scintillators efficiently based on silicon photomultipliers.

An instrument used for measuring multiple scintillators' light output and energy resolution was developed. The instrument consisted of a light sensor array which was composed of 64 discrete SiPMs (Silicon Photomultipliers), a corresponding individual channel readout electronics system, and a data processing algorithm. A Teflon grid and a large interval between adjacent SiPMs were employed to eliminate the optical cross talk among scintillators. The scintillators' light output was obtained by comparing with a reference sample with known light output. Given the SiPM temperature dependency and the difference among each SiPM, a temperature offset correction algorithm and a non-uniformity correction algorithm were added to the instrument. A positioning algorithm, based on nine points, was designed to evaluate the performance of a scintillator array. Tests were performed to evaluate the instrument's performance. The uniformity of 64 channels for light output measurement was better than 98%, the stability was better than 98% when temperature varied from 15 °C to 40 °C, and the nonlinearity under 511 keV was better than 2%. This instrument was capable of selecting scintillators and evaluating the packaging technology of scintillator arrays with high efficiency and accuracy.

The effect of Ag+ on arginine kinase: inhibition kinetics.

We studied the effects of Ag(+) on arginine kinase (AK) from Fenneropenaeus chinensis. Ag(+) inactivated the activity of AK in a dose dependent manner (IC(50) = 15 microM). Kinetic studies showed that the inactivation of AK by Ag(+) was reversible and occurred in a noncompetitive inhibition manner (K(i) = 2.8 microM). Spectroflurorimetry results showed that Ag(+) did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies. However, the secondary structure measured by circular dichroism was slightly changed by Ag(+). Taken together, these data suggest that the active site of AK is flexible, with the complete loss of activity occurring prior to significant changes in overall structures. Our study provides important insight into the inhibitory mechanism of Ag(+) on AK and increases our understanding of the influence of Ag+ on the mechanism of this metabolic enzyme.

The effect of Zn(2+) on human brain creatine kinase: unfolding and aggregation studies.

We studied the effects of Zn(2+) on human brain creatine kinase (HBCK). Zn(2+) inactivated the activity of HBCK in a dose dependent manner (IC50 = 0.06 mM). The time-interval kinetic studies showed that the inactivation followed first-order reaction kinetics with a biphasic process. The spectroflurorimetry results showed that Zn(2+) conspicuously induced the tertiary structural change of HBCK with exposure of its hydrophobic surfaces. On the contrary, the secondary structure was slightly changed by Zn(2+). We also found that HBCK aggregation was induced by Zn(2+). This aggregation was dependent on the temperature and the enzyme and Zn(2+) concentrations. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation and fully recover the conformation and activity of HBCK. Our study provides important insight into the unfavorable effect of Zn(2+) on HBCK and it increases the understanding of the Zn(2+) ligand-binding mechanism to the metabolic brain enzyme.

Activation and inactivation of horseradish peroxidase by cobalt ions.

The effect of cobalt ions (Co2+) on horseradish peroxidase (HRP) was studied in vitro by enzymatic activity assay, electronic absorption spectra, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate(ANS)-binding fluorescence spectra. Co2+ at concentrations below 0.1 mM mildly increased the HRP activity, whereas higher concentrations of Co2+ significantly inactivated HRP in a time and concentration-dependent manner. Steady-state kinetic studies show that Co2+ was a noncompetitive inhibitor of o-dianisidine oxidation by HRP. The Ki value dropped as the incubation time increased. Furthermore, Co2+ was found to be an uncompetitive inhibitor of H2O2. These results suggested that Co2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectroscopic analysis showed that even for high Co2+ concentrations, the structure of HRP as a whole only changed slightly; however, there were significant conformational changes near or in the active site of HRP. Based on the above results, we suggest that Co2+ may bind with some amino acids near or in the active site of HRP and the conformational changes of HRP induced by such binding should be the main reason for activation and inactivation effect of Co2+. The potential binding sites of Co2+ were also proposed.

Persistence of the antihypertensive efficacy of amlodipine and nifedipine GITS after two 'missed doses': a randomised, double-blind comparative trial in Asian patients.

Suboptimal management of hypertension is often a result of poor patient compliance in the form of missed doses of their antihypertensive medication. This multicentre, randomised, double-blind, parallel-group trial was designed to compare the persistence of the antihypertensive efficacy of the amlodipine and nifedipine gastrointestinal therapeutic system (GITS) after two 'missed doses', and also to compare the drugs' overall efficacy and safety in Asian patients with mild-to-moderate essential hypertension. Following a 2-week placebo run-in period, 222 patients were randomised to receive either amlodipine (5 mg daily, increased after 6 weeks if necessary to 10 mg daily, n=109) or nifedipine GITS (30 mg daily, increased after 6 weeks if necessary to 60 mg daily; n=113) for 12 weeks. A placebo was then substituted for further 2 days with continuous ambulatory blood pressure (BP) monitoring. The increases in the last 9 h of mean ambulatory BP on day 2 after treatment withdrawal were significantly less with amlodipine than with nifedipine GITS: 4.4+/-7.0 vs 11.2+/-11.3 mmHg for systolic BP (P

Water soluble polymers in tumor targeted delivery.

The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained. Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.

Preparation and biological evaluation of polymerizable antibody Fab' fragment targeted polymeric drug delivery system.

A new polymerizable antibody Fab' fragment with a PEG spacer (MA-PEG-Fab') was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab' possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab' fragment MA-Fab' with a short spacer. The MA-PEG-Fab' was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce(6) producing an Fab' targeted HPMA copolymer-Mce(6) conjugate. The number and weight average molecular weights of the copolymer were 164000 and 271000 Da, respectively. About two MA-PEG-Fab' fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab' targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate.

Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.

Biorecognizable HPMA copolymer-drug conjugates for colon-specific delivery of 9-aminocamptothecin.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for colon-specific delivery of 9-aminocamptothecin (9-AC) were designed. They hold 9-AC bound via spacers containing amino acid residues and aromatic azo bonds. In vitro release profiles of 9-AC from HPMA copolymer conjugates were evaluated under artificial conditions that simulated large intestinal azoreductase and peptidase activities. The studies indicated that the azo bond was reduced first, followed by the release of unmodified 9-AC from the 9-AC containing fragment by peptidases. Release profiles depended on the chemical structure of the peptide part of the spacer. Conjugates containing leucylalanine showed high colon-specific release of 9-AC when compared to alanine containing conjugates. It appears that the studied conjugates are suitable as colon-specific drug delivery systems.

Modification of cyclosporin A and conjugation of its derivative to HPMA copolymers.

Cyclosporin A (CsA) was epoxidized with m-chloroperoxybenzoic acid in the presence of sodium carbonate or with tert-butyl hydroperoxide in the presence of dioxomolybdenum iminodiethanoxide. The CsA epoxide was not stable and rearranged into a compound with a more stable five-member ring structure. An amino group containing cyclosporin A derivative (CsA amine) was obtained by the reaction of CsA epoxide with excess ethylenediamine. The yield of the CsA amine was 30--40% based on the CsA. An HPMA copolymer--CsA conjugate was prepared by the reaction of the CsA amine with an HPMA and MA-Gly-Phe-Leu-Gly-ONp copolymer. The content of CsA amine in the conjugate was 8.7 wt %. The CsA amine was released from the copolymer by enzymatic hydrolysis with papain.

Differential regulation of the sodium pump alpha-subunit isoform gene by ouabain and digoxin in tissues of rats.

The effects of ouabain and digoxin on both the systolic blood pressure (SBP) and sodium pump alpha-subunit expression in some tissues of rats were compared. Normal rats were injected with ouabain, digoxin, and normal saline (NS), respectively, everyday, and indirect SBP was recorded once a week. Six weeks later, all the rats were killed, and sodium pump alpha1-, alpha2-, and alpha3-subunit mRNA levels were detected in the myocardium, kidney, adrenal gland, aortic smooth muscle, and hypothalamus by the RT-PCR method. The results showed that the SBP of rats infused with ouabain increased significantly at the end of week 6, while no difference in SBP was found between the digoxin and NS groups. The effects of ouabain and digoxin on sodium pump alpha-subunit isoform expression were also different. Myocardium: both ouabain and digoxin stimulated expression of the alpha3-isoform whereas alpha2 was unchanged. Levels of the alpha1 isoform decreased significantly in the ouabain group and decreased slightly in the digoxin group, respectively. Kidney: digoxin had the same effects as ouabain. alpha1 levels increased, but those of alpha2 and alpha3 remained unchanged. Adrenal gland: alpha2 and alpha3 levels increased, but those of alpha1 decreased in the ouabain group. alpha1 and alpha3 levels increased and those of alpha2 remained unchanged in the digoxin group. Aortic smooth muscle: both ouabain and digoxin increased alpha1 and alpha3 expression. alpha2 levels decreased in the digoxin group but remained unchanged in the ouabain group. Hypothalamus: both ouabain and digoxin stimulated alpha1 expression, while alpha2 and alpha3 levels remained unchanged. The results of this study have shown that ouabain and digoxin have the different effects on both the systolic blood pressure and expression of sodium pump alpha-subunit isoforms in some tissues in rats. Further studies on the expression of sodium pump alpha-subunit isoforms might be helpful for the understanding of the physiological role of endogenous ouabain and the molecular mechanisms involved in the pathogenesis of hypertension.

Effects of endogenous ouabain on the development of hypertension in 1k1c hypertensive rats.

This study was designed to evaluate the role of endogenous ouabain (EO) in the development of hypertension in 1ktc (one kidney, one clip) hypertensive rats. First, the EO content of the serum of 1k1c hypertensive rats and normal Sprague-Dawley (SD) rats was detected by the enzyme linked immunosorbent assay method (ELISA). Second, blood pressure changes in the 1k1c rats were recorded directly after the 1k1c rats were injected randomly with anti-ouabain antibody, normal rabbit IgG, and normal saline, respectively. The results showed that EO levels in the serum of 1k1c hypertensive rats were significantly higher than those of normal SD rats (2.25 +/-0.92 microg/l vs. 1.12 +/- 0.17 microg/l, p< 0.01), and correlated significantly with systolic blood pressure (r= 0.59, p< 0.05). Anti-ouabain antibody was able to significantly decrease the blood pressure of 1k1c hypertensive rats in a dose-dependent manner, while normal rabbit IgG or normal saline was not. These results indicate that endogenous ouabain might play an important role in the development of hypertension in 1k1c hypertensive rats.

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates.

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.

Polymerizable Fab' antibody fragments for targeting of anticancer drugs.

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.

In vitro release of 5-fluorouracil with cyclic core dendritic polymer.

This paper describes the first synthesis of a series of dendritic polymers with a core of 1,4,7,10-tetraazacyclododecane. This core was allowed to react with methyl acrylate through a Michael addition and was then amidated with ethylenediamine. Repeating the two steps led to controlled molecular weight increasing and branching on the molecular level and produced four direction poly(amide-amine) dendrimers. We successfully synthesized dendrimers from generation 0. 5 to generation 5.5. Each generation was analyzed by Fourier- transform infrared (FT-IR) spectroscopy, 1H NMR and elemental analysis. Titrimetry was also used to determine the number of -NH2 of each full generation (2.0, 3.0, 4.0, 5.0). SEC (size exclusion chromatography) was performed to test the purity of G-3.0, G-4.0 and G-5.0. Parts of the outer layer -NH2 groups of the dendrimers generation 4 and generation 5 were acylated by acetic anhydride. The solubility in water of the dendrimer was thus greatly enhanced. The acetylated dendrimers were then reacted with 1-bromoacetyl-5-fluorouracil to form dendrimer-5FU conjugates. Hydrolysis of the conjugates in a phosphate buffer solution (pH 7.4) at 37 degreesC will release free 5FU. Different generation of dendrimer-5FU conjugates exert marking influence on the amount of 5FU released. The dendritic polymer seems to be a promising carrier for the controlled release of antitumor drugs.

Functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] for protein modification.

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different functional end groups, namely carboxyl, methyl ester, hydrazide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2'-Azobisisobutyronitrile (AIBN) was used as an initiator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mercaptopropionic hydrazide, and 2-mercaptoethylamine were used as chain-transfer agents. The semitelechelic polymers have been characterized by end-group analysis, size-exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans and the initiator on the molecular weight of the polymers have been investigated. The higher the concentration of mercaptan, the lower the molecular weight of ST-PHPMA. The concentration of initiator did not have a significant effect on the molecular weight of the semitelechelic polymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, alpha-chymotrypsin, has been modified with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu and the conjugates characterized by MALDI-TOF MS. The activity of modified chymotrypsins toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was slightly lower than the activity of the native enzyme. The cleavage of a low molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotrypsins was dependent on their structure. Whereas the activity of the amino group modified chymotrypsins was higher than that of the native enzyme, the activity of carboxyl-modified chymotrypsins was lower than that of the native enzyme. In summary, the data seem to indicate that ST-PHPMA is an effective protein-modifying agent.

Observations on the nature, biosynthesis, secretion and significance of endogenous ouabain.

The human circulation contains four readily distinguishable biologically active inhibitors of the sodium pump that appear to be endogenous to mammals. Of these, one has been purified to homogeneity and by numerous chromatographic, mass spectral, biochemical, and physiological analyses has been shown to be a novel steroidal isomer of ouabain in which the location and orientation of two or more steroidal hydroxyl groups differ. The human endogenous "ouabain" (EO) is a high affinity reversible inhibitor of the pump with inotropic and vasopressor activity. Circulating levels of EO depend upon the adrenal cortex and metabolic events preceding and following pregnenolone formation are involved in EO biosynthesis. Within the adrenal gland, the stimulus-secretion mechanisms for EO secretion are distinct from those for aldosterone highlighting different regulation. Among Caucasians with essential hypertension, 30-45% have elevated circulating levels of EO. Sustained elevation of plasma ouabain in rats induces chronic hypertension with characteristics similar to those in patients and whose severity is determined by inherited factors and renal function. In conclusion, at least one of the mammalian counterparts to the cardiac glycosides is a novel steroidal isomer of ouabain. The isomer is secreted by the adrenal cortex, and augments cardiovascular function. The observation of this entity in the human circulation, the demonstration of its biosynthesis, and the existence of specific receptors suggest to us that EO is a novel adrenocortical hormone and may be part of a broader family of novel mammalian steroids that regulate the sodium pump and other processes.

Angiotensin II stimulates secretion of endogenous ouabain from bovine adrenocortical cells via angiotensin type 2 receptors.

Angiotensin II stimulates secretion of corticosteroids and ouabain-like activity from adrenocortical cells. Distinct adrenocortical angiotensin II receptor subtypes (AT1, AT2) have been described, and the present studies investigated their roles in steroid secretion. Using primary bovine adrenocortical cell cultures under serum free conditions, angiotensin II stimulated the secretions of aldosterone, cortisol, and endogenous ouabain as verified by high-performance chromatography. The dose-response curves for stimulated steroid secretion were parallel with unitary slopes while the half-maximally effective concentrations of angiotensin II were 0.31 to 0.38 nmol/L for secretions of aldosterone and cortisol and 2.3 nmol/L for endogenous ouabain. The nonselective mammalian antagonist (Sar1-Ile8) angiotensin II blocked stimulated secretion of all three steroids without affecting basal output. In the presence of the AT1 antagonist DuP753, angiotensin II-stimulated secretions of aldosterone and cortisol were blocked while secretion of endogenous ouabain was unaffected. In the presence of the AT2 antagonist PD123319, both basal and angiotensin II-stimulated secretions of aldosterone and cortisol were normal while stimulated secretion of endogenous ouabain was inhibited. The secretion of endogenous ouabain was activated maximally by the AT2 agonist CGP42112 under conditions in which aldosterone secretion was unaffected. These results demonstrate that AT2 receptors stimulate secretion of endogenous ouabain from bovine adrenocortical cells. The specificity of AT1 and AT2 receptor stimulation indicates that separate signaling mechanisms having minimal cross talk control the adrenocortical secretions of corticosteroids and cardiac-active steroids. Adrenocortical AT2 receptors may be important in the adaptation to low salt diets and other conditions in which angiotensin II is increased.