[Inhibitory Effects and Mechanisms of Three Benzodiazepines on Helicobacter pylori].

Objective To explore the inhibitory effects and mechanisms of benzodiazepines on Helicobacter pylori (Hp).Methods The Hp international standard strain ATCC43504 was treated with benzodiazepines diazepam,midazolam,and remimazolam,respectively.The treatments with amoxicillin and clarithromycin were taken as the positive controls,and that with water for injection as the negative control.The inhibition zone of each drug was measured by the disk diffusion method.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of each drug against Hp were determined.Hp suspension was configured and treated with diazepam and midazolam,respectively.The bacterial suspension without drug added was used as the control group.The concentration of K+ in each bacterial suspension was measured by an automatic biochemical analyzer before drug intervention(T0)and 1(T1),2(T2),3(T3),4(T4),5(T5),6(T6),and 7 h(T7)after intervention.Hp urease was extracted and treated with 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,2 MIC midazolam,1 mg/ml acetohydroxamic acid,and water for injection,respectively.The time required for the rise from pH 6.8 to pH 7.7 in each group was determined by the phenol red coloring method.Results The inhibition zones of diazepam,midazolam,remimazolam,amoxicillin,clarithromycin,and water for injection against Hp were 52.3,42.7,6.0,72.3,60.8,and 6.0 mm,respectively.Diazepam and midazolam showed the MIC of 12.5 µg/ml and 25.0 µg/ml and the MBC of 25 µg/ml and 50 µg/ml,respectively,to Hp.The concentrations of K+ in the diazepam,midazolam,and control groups increased during T1-T7 compared with those at T0(all P<0.01).The concentration of K+ in diazepam and midazolam groups during T1-T4 was higher than that in the control group(all P<0.01).The time of inhibiting urease activity in the 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,and 2 MIC midazolam groups was(39.86±5.11),(36.52±6.65),(38.58±4.83),(39.25±6.19),(36.36±4.61),and(35.81±6.18)min,respectively,which were shorter than that in the acetohydroxamic acid group(all P<0.01)and had no significance differences from that in the water for injection group(all P>0.05).Conclusion Diazepam and midazolam exerted inhibitory effects on Hp,which may be related to the cleavage of Hp cells rather than inhibiting urease.

ortho-Methoxycarbonylethynylphenyl Thioglycosides (MCEPTs): Versatile Glycosyl Donors Enabled by Electron-Withdrawing Substituents and Catalyzed by Gold(I) or Cu(II) Complexes.

With easily accessible and operator-friendly reagents, shelf-stable ortho-methoxycarbonylethynylphenyl thioglycosides were efficiently prepared. Based on these MCEPT glycoside donors, a novel glycosylation protocol featuring mild and catalytic promotion conditions with Au(I) or Cu(II) complexes, expanded substrate scope encompassing challenging donors and acceptors and clinically used pharmaceuticals, and versatility in various strategies for highly efficient synthesis of glycosides has been established. The practicality of the MCEPT glycosylation protocol was fully exhibited by highly efficient and scalable synthesis of surface polysaccharide subunits of Acinetobacter baumannii via latent-active, reagent-controlled divergent orthogonal one-pot and orthogonal one-pot strategies. The underlying reaction mechanism was investigated systematically through control reactions, leading to the isolation and characterization of the vital catalyst species in MCEPT glycosylation, the benzothiophen-3-yl-gold(I) complex. Based on the results obtained both from control reactions and from studies leading to the glycosylation protocol establishment, an operative mechanism was proposed and the effect of the vital catalyst species reactivity on the results of metal-catalyzed alkyne-containing donor-involved glycosylation was disclosed. Moreover, the mechanism for C-glycosylation side product formation from ortho-(substituted)ethynylphenyl thioglycoside donors with electron-donating substituents was also illuminated.

Identification and quantification analysis of the chemical constituents from Mahonia fortune using Q­Exactive HF Mass Spectrometer and UPLC-ESI-MS/MS.

In this research, a comprehensive and innovative method was established for the qualitative and quantitative analysis of the main components in Mahonia fortune (MF). On the one hand, comprehensive insight of the constituents in MF extracts was achieved with a Q­Exactive HF Mass Spectrometer using data-independent acquisition method. The identification of 17 compounds was based on comparison with authentic reference standards and the deduction of 119 additional compounds both in positive and negative modes was using the MS-dial strategy and comparison with literature data. The proportion of alkaloids and phenols were the most in MF. On the other hand, an ultra-performance liquid chromatographic-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for the quantification of 25 components in MF extract were developed and validated. The method established provided satisfactory precision and accuracy; acceptable recovery and stability; a good linearity and a reasonable limit of detection. The MF samples from 11 different sources were detected, and relative principal component analysis were applied to discriminate these samples. The variations of Columbamine, Jatrorrhizine, Palmatine and Berberine were suggested as important indicators of MF quality. This study supplies a novel and comprehensive method for the quality evaluation of MF. This research presents a MS based analytical strategy which shows an application potential in the analysis of the chemical constituents in Traditional Chinese Medicine (TCM).

Proteomic analysis reveals an impaired Ca2+/AQP5 pathway in the submandibular gland in hypertension.

Hypertension is a systemic disorder that affects numerous physiological processes throughout the body. Improper sodium transport is a common comorbidity of hypertension, and sodium transport is also critical for maintaining the secretion of submandibular glands, whether the function of submandibular glands is affected by hypertension remains unclear. To determine whether hypertension induces changes in the protein expression of submandibular glands, we compared the proteome of submandibular glands from 14-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats using LC-MS/MS. The results revealed that 95 proteins displayed different levels of expression between the submandibular glands from the SHRs and WKYs. Among these, 35 proteins were more abundant, and 60 proteins were less abundant in the SHR compared with the WKY rats. Specifically, aquaporin 5 and parvalbumin, which are correlated with water transport and intracellular Ca2+ signal transduction, were verified to exhibit differences in protein abundance. Impaired Ca2+ response to carbachol was confirmed in the acinar cells from SHRs, and hyposecretion by the submandibular glands was further confirmed by in vivo saliva collection. In conclusion, the proteomic analysis of the submandibular glands of SHRs revealed novel changes in protein abundance that provides possible mechanisms connecting hypertension and hyposecretion in submandibular glands.

Expression and significance of VEGF and p53 in degenerate intervertebral disc tissue.

OBJECTIVE: To investigate the mechanism of expression and significance of vascular endothelial growth factor (VEGF) and p53 in degenerate intervertebral disc tissue. METHODS: Pathological sections collected from 156 patients with lumbar disc herniation after surgery were tested by immunohistochemistry method, for evaluation of the expression of VEGF and p53 in degenerate intervertebral disc tissue. RESULTS: 98 cases (62.8%) with vascular infiltration phenomenon are found, and positive rates of VEGF and p53 in degenerate intervertebral disc tissue are 73.42% (116/156) and 58.97% (92/156); co-expression rate is 53.2%(83/156); the expression rates of VEFG and p53 are significantly higher in the tissue with blood vessel infiltration than in the tissue without infiltration; there is a close relationship of VEGF with p53. CONCLUSIONS: VEGF and p53 gene synergetic express in degenerate intervertebral disc tissue, working together in neovascularization and infiltration, and accelerating intervertebral disc tissue degeneration.

Roles of vimentin and 14-3-3 zeta/delta in the inhibitory effects of heparin on PC-3M cell proliferation and B16-F10-luc-G5 cells metastasis.

AIM: To investigate the inhibitory effects of heparin on PC-3M cells proliferation in vitro and B16-F10-luc-G5 cells metastasis in Balb/c nude mice and identify the protein expression patterns to elucidate the action mechanism of heparin. METHODS: Human prostate cancer PC-3M cells were incubated with heparin 0.5 to 125 µg/mL for 24 h. The proliferation of PC-3M cells was assessed by MTS assay. BrdU incoporation and Ki67 expression were detected using a high content screening (HCS) assay. The cell cycle and apoptosis of PC-3M cells were tested by flow cytometry. B16-F10-luc-G5 cardinoma cells were injected into the lateral tail vein of 6-week old male Balb/c nude mice and heparin 30 mg/kg was administered iv 30 min before and 24 h after injection. The metasis of B16-F10-luc-G5 cells was detected by bioluminescence assay. Activated partial thromboplastin time (APTT) and hemorheological parameters were measured on d 14 after injection of B16-F10-luc-G5 carcinoma cells in Balb/c mice. The global protein changes in PC-3M cells and frozen lung tissues from mice burdened with B16-F10-luc-G5 cells were determined by 2-dimensional gel electrophoresis and image analysis. The protein expression of vimentin and 14-3-3 zeta/delta was measured by Western blot. The mRNA transcription of vimentin, transforming growth factor (TGF)-ß, E-cadherin, and α(v)-integrin was measured by RT-PCR. RESULTS: Heparin 25 and 125 µg/mL significantly inhibited the proliferation, arrested the cells in G(1) phase, and suppressed BrdU incorporation and Ki67 expression in PC-3M cells compared with the model group. But it had no significant effect on apoptosis of PC-3M cells. Heparin 30 mg/kg markedly inhibits the metastasis of B16-F10-luc-G5 cells on day 8. Additionally, heparin administration maintained relatively normal red blood hematocrit but had no influence on APTT in nude mice burdened with B16-F10-luc-G5 cells. Thirty of down-regulated protein spots were identified after heparin treatment, many of which are related to tumor development, extracellular signaling, energy metabolism, and cellular proliferation. Vimentin and 14-3-3 zeta/delta were identified in common in PC-3M cells and the lungs of mice bearing B16-F10-luc-G5 carcinoma cells. Heparin 25 and 125 µg/mL decreased the protein expression of vimentin and 14-3-3 zeta/delta and the mRNA expression of α(v)-integrin. Heparin 125 µg/mL decreased vimentin and E-cadherin mRNA transcription while increased TGF-ß mRNA transcription in the PC-3M cells, but the differences were not significant. Transfection of vimentin-targeted siRNA for 48 h significantly decreased the BrdU incoporation and Ki67 expression in PC-3M cells. CONCLUSION: Heparin inhibited PC-3M cell proliferation in vitro and B16-F10-luc-G5 cells metastasis in nude mice by inhibition of vimentin, 14-3-3 zeta/delta, and α(v)-integrin expression.

[A comparison of proteomic analysis of Helicobacter pylori in patients with gastritis and gastric cancer between areas of high and low incidence of gastric cancer].

OBJECTIVE: To identify the differentially expressed proteins of Helicobacter pylori (Hp) in patients with gastritis and gastric cancer from areas of high and low incidence of gastric cancer by 2-dimensional electrophoresis (2-DE), and to discuss the role of bacterial factor in pathogenesis. METHODS: Hp in the endoscopic biopsy specimens of gastric mucosa of patients with gastritis and gastric cancer from areas of high (Xining) and low (Beijing) incidence of gastric cancer, were separated, cultured and saved at -80°C. The bacteria were recovered. Then the whole-cell protein of the Hp were extracted and characterized by 2-DE. The different protein spots were analyzed by PDQuest analysis software and identified by electrospray ionization quadruple time-of-flight mass spectrometry (ESI-Q-TOF-MS), and searched by the Mascot database. RESULTS: Nine differentially expressed proteins were identified, and four protein spots were over expressed in the protein maps from gastric cancer in both areas, which were: Urease subunit alpha, chaperone protein dnaK, superoxide dismutase, DNA-directed RNA polymerase subunit alpha; two protein spots were over expressed in the protein maps from gastritis in both areas, which were: Probablethiol peroxidase, nucleoside diphosphate kinase; 60×10(3) chaperonin, and inorganic pyrophosphatase were over expressed only in the protein map from gastric cancer in Xining; S-ribosyl homocysteinelyase was over expressed only in the protein map from gastric cancer in Beijing. CONCLUSION: There are differences between proteomic analyses of Hp in patients with gastritis and gastric cancer in areas of high and low incidents of gastric cancer, but 2/3 of the protein spots over expressed in the areas are consistent. The protein spots over expressed from gastric cancer in the area with high incidence of gastric cancer are more than in the area with low incidence of gastric cancer. For the Hp extracted from patients with gastric cancer, the mechanism of gastric cancer may be similar, but the role of the Hp from the area with high incidence of gastric cancer may be stronger.

Comparative proteome analysis of Helicobacter pylori clinical strains by two-dimensional gel electrophoresis.

OBJECTIVE: To investigate the pathogenic properties of Helicobacter pylori by comparing the proteome map of H. pylori clinical strains. METHODS: Two wild-type H. pylori strains, YN8 (isolated from biopsy tissue of a gastric cancer patient) and YN14 (isolated from biopsy tissue of a gastritis and duodenal ulcer patient), were used. Proteomic analysis, using a pH range of 3-10 and 5-8, was performed. The individual proteins were identified by quadrupole time-of-flight (Q-TOF) mass spectrometer and protein database search. RESULTS: Variation in spot patterns directed towards differential protein expression levels was observed between the strains. The gel revealed prominent proteins with several protein "families". The comparison of protein expressions of the two strains reveals a high variability. Differentially present or absent spots were observed. Nine differentially expressed protein spots identified by Q-TOF included adenosine triphosphate (ATP)-binding protein, disulfide oxidoreductase B (DsbB)-like protein, N utilization substance A (NusA), ATP-dependent protease binding subunit/heat shock protein, hydantoin utilization protein A, seryl-tRNA synthetase, molybdenum ABC transporter ModD, and hypothetical proteins. CONCLUSIONS: This study suggests that H. pylori strains express/repress protein variation, not only in terms of the virulence proteins, but also in terms of physiological proteins, when they infect a human host. The difference of protein expression levels between H. pylori strains isolated from gastric cancer and gastritis may be the initiator of inflammation, and result in the different clinical presentation. In this preliminary study, we report seven differential proteins between strains, with molecule weights from approximately 10 kDa to approximately 40 kDa. Further studies are needed to investigate those proteins and their function associated with H. pylori colonization and adaptation to host environment stress.

[Therapeutic effect of electroacupuncture on chronic orchialgia].

OBJECTIVE: To compare the efficiency difference in chronic orchialgia between electroacupuncture and western medicine and explore the better therapy for chronic orchialgia. METHODS: Eighty-six cases of chronic orchialgia were randomly divided into an electroacupuncture group (56 cases) and an Indomethacin group (30 cases). Except the conventional oral administration of Amitriptyline for anti-depression, electroacupuncture was applied at Shenshu (BL 23), Ciliao (BL 32), Sanyinjiao (SP 6), Taichong (LR 3) and others in electroacupuncture group, and Indometacin suppository anal therapy was used in Indometacin group. The session of treatment in two groups was 2 weeks. Before and after treatment, pain number assessment scale was adopted for the assessment of pain severity separately to compare the differences in pain score on 3rd, 7th, 10th and 14th days after treatment. RESULTS: The pain score at each time point was improved remarkably as compared with that before treatment in two groups (P < 0.05, P < 0.01). In comparison on the 7th, 10th and 14th days of two groups, the pain scores in electroacupuncture group was lower than those in Indometacin group (all P < 0.05). CONCLUSION: Electroacupuncture combined with Amitriptyline treatment is quite effective on chronic orchialgia, which is superior to the effect of Indo methacin combined with Amitriptyline treatment and deserves to be promoted in application.

[Two types of MWNTs with different surface modifications induce differential expression of proteins in RAW264.7 cells].

OBJECTIVE: To compare the different expression of protein in RAW264.7 macrophage cells induced by two types of MWNTs (multi-walled carbon nanotubes) with different surface modifications (acid-treated MWNTs and tau-MWNTs modified by taurine). METHODS: Treating cells with both types of MWNTs in 20 mg/L and 24 h, with a blank-control group set. Cells are lysed by using urea and by ultrasonicating in ice bath, then total proteins of cells are extracted. Using two-dimensional gel electrophoresis to separate total proteins of cells, searching for the differential expressed protein spots on the images of the gels with silver staining. Identifying the differentially expressed proteins via mass spectrometry, and studying the mechanism of effects on cells imposed by two types of MWNTs at protein level. RESULTS: There are 13 spots of protein with notably differential expression among three treated groups (including blank controls). Their functions involve apoptosis-related, calcium-binding, cell-cycle related, DNA synthesis, folding of proteins, and energy metabolism, etc. The results are consistent with our previous studies about the cytotoxicity of Both types of MWNTs including induction of apoptosis and mitochodira damage. CONCLUSION: Both two types of MWNTs could induce alteration of protein expression in RAW264.7 cells. With different surface modifications, they imposed different effects. High throughout proteomics could be applied in toxicity assessment and mechanism investigation about carbon nanotubes.

Proteome analysis of the sera from Chinese Parkinson's disease patients.

Clinical proteomics is a powerful tool that can be used to identify proteins that are differentially expressed in disease states, leading to greater understanding of the molecular and cellular events that contribute to disease. The aim of this study was to identify protein changes in the sera from Chinese Parkinson's disease (PD) patients, with the goal of finding biomarkers for PD diagnosis, and to elucidate the events occurring at the onset of PD. Using differential display to identify proteins with altered expression in PD patients, we obtained 15 protein spots corresponding to 13 different gene products that were likely to be involved in PD. Two-dimensional gel electrophoresis and mass spectrometry were used to identify differentially expressed proteins, 7 of which have never previously been associated with PD patients. They are likely to be involved in antioxidation, lipid metabolism, intracellular transport, cell proliferation and immunoregulation. The altered levels of these proteins may be related to the pathophysiological mechanisms of PD. As a result, some of these proteins could be considered as candidate biomarkers.

Effects of chronic morphine treatment on protein expression in rat dorsal root ganglia.

The initial aim of this study was to identify protein changes in the dorsal root ganglia (DRG) associated with long-term morphine treatment. We carried out a differential proteomics analysis on samples from the DRG of control and morphine-dependent rats (5-40 mg/kg, subcutaneously, twice daily for 28 days) after 4 days of morphine withdrawal. Proteins showing a statistically significant variation between the two groups were selected for identification by mass spectrometric analysis. Twelve proteins were unambiguously identified, with the majority being enzymes involved in energy metabolism and protein degradation, signaling and cytoskeletal proteins. Aldolase C and proteasome subunit alpha type 3 (PRC8 or alpha7) were further examined by Western blot analysis in the DRG, prefrontal cortex, nucleus accumbens, striatum, hippocampus, ventral tegmental area and locus coeruleus. In addition, expression of PRC8 in the nucleus accumbens tissue after spontaneous or naloxone-precipitated withdrawal was determined using immunohistochemical staining. Our results indicate that the expression levels of aldolase C and PRC8 proteins were altered in a time- and region-specific manner, and suggest that they could be implicated in the molecular changes underlying adaptations of neurons observed with chronic morphine treatment.

Serum amyloid A mediates the inhibitory effect of Ganoderma lucidum polysaccharides on tumor cell adhesion to endothelial cells.

Ganoderma ludicum polysaccharides (GlPS) are the major bioactive composition of Ganoderma lucidum, a well-recognized oriental medical fungus. The published data have shown a complementary effect of GlPS in cancer therapy. The present study was designed to determine the anti-tumor efficacy of GlPS and the possible mechanism covering this effect. Murine Sarcoma 180 (S180) model was established, and GlPS administered orally for 10 days. On the 10th day, tumors were weighed to assess the inhibitory effect of GlPS and sera were collected for proteomic analysis and in vitro study. The in vivo results demonstrated that 25, 50 and 100 mg/kg GlPS inhibited S180 growth by 32.67, 44.80 and 45.24%, respectively (P<0.01). Proteomic study revealed marked protein changes after the process of treatment. Three significantly changed proteins were identified by ESI-Q-TOF-MS and database search indicated that they were haptoblobin, apolipoprotein A-II and serum amyloid A (SAA), respectively. Additionally, the expression change of SAA was confirmed by both Western blot and RT-PCR. The adhesion assay showed that GlPS-treated sera dramatically inhibited the adhesion ability of human prostate carcinoma (PC-3M) cells to human umbilical cord vascular endothelial cells (HUVECs), and this effect partially recovered after immunodepletion by the antibody against SAA. Collectively, these results suggest that GlPS inhibited the tumor growth and tumor cell adhesion to HUVECs via up-regulation of SAA protein expression.

[Difference analysis on proteome of Helicobacter pylori in patients with peptic ulcer, gastritis, and gastric cancer].

OBJECTIVE: To identify the different proteins of Helicobacter pylori (H. pylori) in gastric cancer, peptic ulcer, and gastritis initially. METHODS: H. pylori in the endoscopic biopsy specimens of gastric mucosa of patients with gastric cancer, peptic ulcer, or gastritis, 3 specimens for each disease, were separated and cultured. The whole-cell protein of the H. pylori was extracted by lysis buffer and sonication. The protein concentration of the bacteria cell lysates was measured by the Bradford method. The protein maps of H. pylori were obtained by two-dimensional gel electrophoresis (2-DE) and the different proteins in gastric cancer, peptic ulcer and gastritis were analyzed by Image Master v 5.0. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) were performed to identify the different proteins. The differential proteins were searched by the Mascot database at www.matrixscience.com. RESULTS: Four protein spots of H. pylori were over-expressed in the protein maps from gastric cancer in comparison with those from peptic ulcer and gastritis. Mass identification showed that the 4 proteins were thioredoxin, adenylate kinase, single-stranded DNA-binding protein, and ribosomal protein 50S L7/L12, with the Mowse scores of 94, 286, 139 and 132, and with the sequence coverage rates of 77%, 33%, 33%, and 28% respectively. CONCLUSION: Anti-oxidant and inhibiting apoptosis, thioredoxin may be related to gastric carcinogenesis induced by H. pylori. Proteomics technology has a widespread perspective in the field of relationship between pathogenic bacteria and gastric cancer.