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Background Pleomorphic adenoma (PA) is the most common benign tumor that occurs in the salivary glands; however, tracheobronchial PA is rarely observed. To the best of our knowledge, fewer than 50 cases have been reported in the literature. We report a 49-year-old woman who had been treated for asthma for 2 years before being diagnosed with PA of the trachea. Case summary A 49-year-old woman was referred to our hospital due to dyspnea upon exertion and chronic cough with wheezing for 2 years. Laboratory tests showed an elevated white blood cell count, absolute neutrophil count, and percentage of neutrophils. A chest computerized tomography scan showed a well-defined, soft-tissue density lesion measuring 2.4 cm × 2.1 cm in the lower trachea. Flexible bronchoscopy revealed that nearly 90% of the tracheal lumen was obstructed. The histopathological and immunohistochemistry features suggested PA of the trachea. Furthermore, we review the characteristics of 29 patients with tracheobronchial PA over the last 30 years. Conclusion Tracheobronchial PA occurs without gender predominance, mostly in the lower or upper trachea, and has a low recurrence rate. The median age at diagnosis is 48 years. The most common symptoms are cough, stridor, dyspnea, and wheezing.

Fluorouracil (5-FU) and oxaliplatin (L-OHP) are the most commonly used chemotherapy drugs for colorectal cancer, though resistance is common. Compound Sophora injection is a traditional Chinese medicine that can protect the liver against oxidation, improve immunity, and enhance sensitivity to chemotherapy; it may have an effect of reversing resistance in 5-FU- and L-OHP-resistant gastric cancer cells (5-FU/SW480 and L-OHP/SW480, respectively). A concentration gradient experiment was performed to identify a nontoxic dose of compound Sophora injection. 5-FU/SW480 and L-OHP/SW480 cells were treated with the nontoxic dose of compound radix Sophorae injection for 48 h, and changes in drug resistance to 5-FU and L-OHP were detected. Alterations in apoptosis and the cell cycle were assessed, as were the mRNA and protein levels of permeability glycoprotein (P-gp), annexin A1 (ANXA1), and ATP-binding cassette superfamily G member 2 (ABCG2). Flow cytometry showed a reduction in the number of cells in the G1 phase and an increase of cells in the S phase (P<0.05). mRNA and protein expression of P-gp and ABCG2 was significantly higher in 5-FU/SW480 and L-OHP/SW480 cell lines, and ANXA1 expression decreased significantly (P<0.05). Compound Sophora injection can reverse the drug resistance of 5-FU/SW480 and L-OHP/SW480 cell lines to 5-FU and L-OHP, respectively, possibly through a mechanism involving reduced expression of P-gp and ABCG2 but enhanced expression of ANXA1, which is the basis for the identification of clinical drug resistance in colorectal cancer.

Purpose To evaluate the value of impulse oscillometry (IOS) and quantitative HRCT parameters for differentiating asthma-COPD overlap (ACO) in COPD patients. Patients and Methods We enrolled 44 controls and 66 COPD patients, divided into the pure COPD group (n=40) and the ACO group (n=26). Spearman correlation analysis was utilized to assess the relationship between the quantitative HRCT and IOS parameters. A binary logistic regression analysis was performed to analyze the associations between the different variables and the risk of ACO. Receiver operating characteristic (ROC) curves were employed to identify the optimal cutoff and assess the diagnostic value of relative volume change -856 HU to -950 HU (RVC-856 to -950), decrease in the resistance from 5 Hz to 20 Hz (R5-R20) and their combination in predicting ACO. Bootstrapping validation was used to evaluate the internal validation. The concordance index (C-index) and calibration plot were calculated to assess the discrimination and calibration of the prediction model. Results Binary logistic regression analysis indicated that RVC-856 to -950 and the IOS parameters (R5-R20, R5, X5) were independently correlated with a higher risk of developing ACO after adjusting for age, sex, body mass index (BMI), history of smoking, exacerbation and atopy or allergic rhinitis. A correlation analysis showed a good correlation between the pulmonary function parameters and RVC-856 to -950, with a weaker correlation with the % area of low attenuation (LAA%) in ACO patients. Combining RVC-856 to -950 and R5-R20 to predict ACO, the AUC was 0.909, and the optimal cutoff value was >-0.62 for RVC-856 to -950 and >0.09 for R5-R20. Good calibration and favorable discrimination were displayed with a higher C-index. Conclusion More serious small airway impairment exists in ACO patients. The combination of RVC-856 to -950 and R5-R20 could be applied to differentiate ACO from COPD patients.

Background: Proton pump inhibitors (PPIs) are validated gastric acid suppressors and have been widely used to treat patients with active duodenal ulcers. Although existing PPIs have shown great efficacy, many scientists are still devoted to developing more effective PPIs with better safety profile. Herein, we aimed to compare the safety and efficacy of anaprazole in duodenal mucosal healing, a novel PPI, to that of rabeprazole.Methods: In this multicenter, randomized, positive-controlled, double-blinded, parallel-group phase II clinical trial, a total of 150 qualified patients with endoscopically confirmed active duodenal ulcers were randomized (1:1:1) to receive rabeprazole 10 mg, anaprazole 20 mg or anaprazole 40 mg for 4 weeks. The ulcer healing rates after 4 weeks of treatment were compared between groups by independent central review and investigator review. In addition, symptoms and safety were evaluated.Results: Based on the independent central review, the ulcer healing rates of the 10 mg rabeprazole, 20 mg anaprazole and 40 mg anaprazole groups were 88.0, 85.1, and 87.5%, respectively, in the FAS population and 88.9, 86.0, and 90.9%, respectively, in the PPS population. The ulcer healing rate difference between anaprazole 20 mg and Rabeprazole 10 mg is −2.9% (95% CI, −16.5–10.7%), and −0.5% (95% CI, −13.5–12.5%) between anaprazole 40 mg and Rabeprazole 10 mg, in the FAS population. Based on the investigator review, the ulcer healing rates of the 10 mg rabeprazole, 20 mg anaprazole, and 40 mg anaprazole groups were 72.0, 70.2, and 77.1%, respectively, in the FAS population and 75.6, 72.1, and 79.5%, respectively, in the PPS population. The ulcer healing rate difference between anaprazole 20 mg and Rabeprazole 10 mg is −1.8% (95% CI, −19.8–16.3%), and 5.1% (95% CI, −12.2–22.3%) between anaprazole 40 mg and Rabeprazole 10 mg, in the FAS population. Most patients (>90%) eventually achieved complete symptom relief. The incidence rates of adverse events were of no significant differences among the treatment groups. Potential possible better liver tolerance was observed in two anaprazole dose groups than rabeprazole 10 mg group.Conclusion: Both at a dosage of 20 and 40 mg daily, anaprazole, is effective with good safety profile in the treatment of active duodenal ulcers in this Phase 2 study, which allows anaprazole to be advanced to a phase III clinical trial.Clinical Trial Registration:https://www.clinicaltrials.gov/ct2/results?cond=&term=NCT04503629&cntry=&state=&city=&dist=, Identifier: CTR20181464, NCT04503629.

Matrine may be protective against colorectal cancer (CRC), but how it may work is unclear. Thus, we explored the underlying mechanisms of matrine in CRC. Matrine-related proteins and CRC-related genes and therapeutic targets of matrine in CRC were predicted using a network pharmacology approach. Five targets, including interleukin 6 (IL-6), the 26S proteasome, tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-β1) and p53, and corresponding high-mobility group box 1 (HMGB1) signaling and T helper cell differentiation were thought to be associated with matrine’s mechanism. Expression of predicted serum targets were verified in a 1,2-dimethylhydrazine dihydrochloride-induced CRC model rats that were treated with matrine (ip) for 18 weeks. Data show that matrine suppressed CRC growth and decreased previously elevated expression of IL-6, TNF-α, p53, and HMGB1. Matrine may have had a therapeutic effect on CRC via inhibition of HMGB1 signaling, and this occurred through downregulation of IL-6, TNF-α, and HMGB1.

Guan-Sheng Zeng,* Li-Chang Chen,* Hui-Zhen Fan, Ling-Ling Wu, Xi-Ping Wu, Ze-Kui Fang, Xi He, Hua-Peng Yu Department of Pulmonary and Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China *These authors contributed equally to this work Background and objective: The distance of 6-minute walk test (D6MWT) has been widely used in the assessment of functional status in patients with COPD, while very little attention has been paid to the role of steps of 6-minute walk test (S6MWT). The purpose of this study was to investigate the relationship between S6MWT and other physiologic parameters of COPD. Patients and methods: Seventy patients with stable COPD were enrolled consecutively in this cross-sectional study. Pulmonary function tests, including spirometry, impulse oscillometry (IOS) and the single-breath diffusing capacity of the lungs for carbon monoxide (DLCO), were carried out at rest. Quality of life was assessed by health-related quality of life (HRQoL) questionnaires, including modified Medical Research Council dyspnea scale (mMRC), St George’s Respiratory Questionnaire, Chronic Obstructive Pulmonary Disease Assessment Test (CAT) and Clinical Chronic Obstructive Pulmonary Questionnaire. Both steps and distance were measured in the following 6-minute walk test (6MWT). Results: Both S6MWT and D6MWT showed significant correlation with spirometry, IOS, DLCO parameters and HRQoL questionnaires score. Both pre- and post-6MWT inspiratory capacity showed significant correlation with S6MWT (ρ=0.338, P=0.004; ρ=0.359, P=0.002, respectively), whereas did not correlate with D6MWT (ρ=0.145, P=0.230; ρ=0.160, P=0.189, respectively). In stepwise multiple regression analysis, mMRC grade, age and CAT score remained as significant predictors in the final model for D6MWT (adjusted R2=0.445, P

The turmeric derivative curcumin protects against cerebral ischemic injury. We previously demonstrated that curcumin activates peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-activated transcription factor involved in both neuroprotective and anti-inflammatory signaling pathways. This study tested whether the neuroprotective effects of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury of rat cortical neurons are mediated (at least in part) by PPARγ. Curcumin (10 μM) potently enhanced PPARγ expression and transcriptional activity following OGD/R. In addition, curcumin markedly increased neuronal viability, as evidenced by decreased lactate dehydrogenase release and reduced nitric oxide production, caspase-3 activity, and apoptosis. These protective effects were suppressed by coadministration of the PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662) and by prior transfection of a small-interfering RNA (siRNA) targeting PPARγ, treatments that had no toxic effects on healthy neurons. Curcumin reduced OGD/R-induced accumulation of reactive oxygen species and inhibited the mitochondrial apoptosis pathway, as indicated by reduced release of cytochrome c and apoptosis-inducing factor and maintenance of both the mitochondrial membrane potential and the Bax/Bcl-2 ratio. Again, GW9662 or PPARγ siRNA transfection mitigated the protective effects of curcumin on mitochondrial function. Curcumin suppressed IκB kinase phosphorylation and IκB degradation, thereby inhibiting nuclear factor-κ B (NF-κB) nuclear translocation, effects also blocked by GW9662 or PPARγ siRNA. Immunoprecipitation experiments revealed that PPARγ interacted with NF-κB p65 and inhibited NF-κB activation. The present study provides strong evidence that at least some of the neuroprotective effects of curcumin against OGD/R are mediated by PPARγ activation.

The turmeric derivative curcumin protects against cerebral ischemic injury. We previously demonstrated that curcumin activates peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-activated transcription factor involved in both neuroprotective and anti-inflammatory signaling pathways. This study tested whether the neuroprotective effects of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury of rat cortical neurons are mediated (at least in part) by PPARγ. Curcumin (10 μM) potently enhanced PPARγ expression and transcriptional activity following OGD/R. In addition, curcumin markedly increased neuronal viability, as evidenced by decreased lactate dehydrogenase release and reduced nitric oxide production, caspase-3 activity, and apoptosis. These protective effects were suppressed by coadministration of the PPARγ antagonist 2-chloro-5-nitrobenzanilide (GW9662) and by prior transfection of a small-interfering RNA (siRNA) targeting PPARγ, treatments that had no toxic effects on healthy neurons. Curcumin reduced OGD/R-induced accumulation of reactive oxygen species and inhibited the mitochondrial apoptosis pathway, as indicated by reduced release of cytochrome c and apoptosis-inducing factor and maintenance of both the mitochondrial membrane potential and the Bax/Bcl-2 ratio. Again, GW9662 or PPARγ siRNA transfection mitigated the protective effects of curcumin on mitochondrial function. Curcumin suppressed IκB kinase phosphorylation and IκB degradation, thereby inhibiting nuclear factor-κ B (NF-κB) nuclear translocation, effects also blocked by GW9662 or PPARγ siRNA. Immunoprecipitation experiments revealed that PPARγ interacted with NF-κB p65 and inhibited NF-κB activation. The present study provides strong evidence that at least some of the neuroprotective effects of curcumin against OGD/R are mediated by PPARγ activation.

To explore the relationship of vascular endothelial growth factor C (VEGF-C) and collagen triple helix repeat containing 1 (CTHRC1) expression with the carcinogenesis and prognosis of rectal cancer.Cancer tissue samples from 120 rectal cancer patients confirmed by pathology in the People's Hospital of Yichun City from September 2005 to September 2010 were included in the study. Expressions of CTHRC1 and VEGF-C were examined by immunohistochemistry and their correlations with clinicopathological features and prognosis were analyzed.The expression of VEGF-C was positively correlated with tumor size (r=0.943), TNM stages (r=0.784) and tumor differentiation (r=0.773) (all P0.05). Similarly, the expression of CTRHC1 was also positively correlated with tumor size (r=0.829), TNM stages (r=0.632) and tumor differentiation (r=0.532) (all P0.05). Rectal cancer patients with low expression of VEGF-C and CTHRC1 had significantly longer survival than those with high expression of VEGF-C and CTHRC1 [(40.0±1.3) vs. (35.4±0.5) months, P0.01, (39.0±0.5) vs. (35.0±0.5) months, P=0.014].VEGF-C and CTHRC1 may synergistically promote the invasion and metastasis of human rectal cancer, and provide evidence in predicting the prognosis of patients.

To explore the effect and mechanism of lipopolysaccharides (LPS)-induced CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs) on airway inflammation in asthmatic mice.A total of 34 female BALB/c mice were selected. Among them, 4 mice received an intraperitoneal injection of LPS for inducing the accumulation of MDSCs. And the MDSCs were separated with CD11b immunomagnetic beads from spleen extract. Another 30 mice were randomly divided into normal control, asthmatic and cell treatment groups. The mice in the asthmatic and cell treatment groups were sensitized with ovalbumin by a combination of intraperitoneal injection and challenges to establish the murine asthmatic model. At Days 14 and 21 post-sensitization, the mice in cell treatment group received an intravenous injection of LPS-induced MDSCs. At 24 hours after the last allergen challenge, the number of inflammatory cells were counted and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) was performed to analyze the degree of airway inflammation in conjunctions with pathological sections. The BALF and serum levels of interleukin-13 were measured by enzyme-linked immunosorbent assay (ELISA). The number of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in peripheral blood was measured by flow cytometry.The total number of cells, the percentage of neutrophils and eosinophils of BALF in the cell treatment group [(17.0 ± 8.3)×10⁴/ml, 11.1% ± 2.0%, 9.8% ± 2.9%] were significantly lower than those in the asthmatic group [(36.0 ± 15.9)×10⁴/ml, 20.8% ± 4.0%, 14.1% ± 4.2%] (P = 0.000, 0.000, 0.011). Compared to the asthmatic group, the BALF and serum levels of IL-13 were significantly lower [(34.7 ± 7.1) vs (105.0 ± 9.0) ng/L, (34.0 ± 4.7) vs (48.1 ± 6.1) ng/L] (both P = 0.000) and the number of CD4⁺CD25⁺Foxp3⁺ regulatory T cells increased in peripheral blood (8.0% ± 1.3% vs 5.1% ± 2.1%, P = 0.002) and airway inflammation was significantly relieved in the cell treatment group.LPS-induced MDSCs may improve airway inflammation through up-regulating Tregs in peripheral blood and suppressing Th2 effector function in asthmatic mice.

Fish-eye disease (FED) and familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) are rare disorders of lipid metabolism linked to mutations in the LCAT gene. Eleven LCAT cDNA constructs associated with FED and FLD were prepared by site-directed mutagenesis and expressed in COS-6 cells. Analysis of total RNA from wild-type, FED, and FLD transfectants revealed that all contained LCAT-specific mRNA. Western blot analysis demonstrated that all LCAT transfectants synthesized LCAT. Mean LCAT secretion by FED transfectants was slightly lower than secretion by wild-type transfectants, whereas secretion by FLD transfectants was much lower. The specific activities of FED and FLD LCAT against model high density lipoproteins were 6% and 11%, respectively, of wild-type activity. The ratios of the LCAT activities against low density lipoproteins to those against model high density lipoproteins decreased in the order FED mutants > FLD mutants approximately wild type. FED and FLD LCAT mutants are different: the former are more active against low density lipoproteins, and the latter are less secretion-competent. The greater reactivity of FED LCAT against low density lipoproteins may explain the relative mildness of the clinical manifestations of FED compared to those of FLD.

To explore the effects of lipopolysaccharide (LPS)-induced myeloid-derived suppressor cells (MDSCs) on the proliferation of spleen T lymphocytes.BALB/c mice were randomly divided into two groups: LPS group and normal control group. They were injected intraperitoneally with LPS and normal saline solution respectively. MDSCs were separated with CD11b immunomagnetic beads from the spleen extract of mice. The morphological characteristics of MDCSs were observed by Wright-Giemsa staining and the characteristic molecules on cell surface identified by flow cytometry. And the effects of MDSCs on the in vitro proliferation of T cells were determined by methyl-thiazolyl-tetrazolium bromide (MTT).The proportion of MDSCs in the spleen of the LPS group was much more than that of the normal control group (27.4% ± 6.6% vs 5.1% ± 3.8%; t = 5.06, P = 0.007). CD11b(+)Gr-1(+)MDSCs could be separated by CD11b immunomagnetic beads from the spleen of mice injected with LPS at a high purity of 84.0% ± 4.2%. MTT method showed that the proliferation of T cells decreased significantly after a co-cultivation with CD11b(+)MDSCs versus the control group. And it was positively correlated with the number of MDSCs (F = 46.26, P = 0.000).A high purity of LPS-induced myeloid-derived suppressor cells may be separated with CD11b immunomagnetic beads. And it has dose-dependent inhibitory effects on the proliferation of the spleen T lymphocytes.

To explore whether or not CD8(+)CD28(-)T cell play a pathogenic role in asthma and detect the effects of dexamethasone (DXM).A total of 30 mice were randomly divided into 3 groups: asthmatic group, DXM group and control group (n = 10 each). The asthmatic and DXM groups were sensitized twice and inhaled ovalbumin. The DXM Group received an intraperitoneal injection of DXM 1mg/kg before inhaling ovalbumin. After successful modeling, 3 mice were selected randomly from each group to measure the airway responsiveness. Also a bronchoalveolar lavage cytological study was performed and lung tissue sections were prepared for histopathologic examination to evaluate the airway inflammation. The content of IgE in bronchoalveolar lavage fluid (BALF) was detected with a murine IgE ELISA kit. And the fractions of CD8(+)CD28(-)T cell of peripheral blood and BALF were tested by flow cytometry to analyze the correlation between IgE, eosinophils (EOS) of BALF and CD8(+)CD28(-)T cell of blood.The airway hyperresponsiveness in asthmatic and DXM groups were significantly higher than that in the control group. The number of total cells and EOS of BALF in the asthmatic group [(5.56 ± 4.06) × 10(2)/L; (3.29 ± 2.23) × 10(2)/L] were significantly higher than that in control group [(0.91 ± 0.65) × 10(2)/L, P = 0.003; (0.43 ± 0.37) × 10(2)/L, P = 0.001] and DXM group [(2.59 ± 1.69) × 10(2)/L, P = 0.044; (1.11 ± 0.73) × 10(2)/L, P = 0.008]; while the DXM group was insignificantly higher than the control group (P = 0.234, P = 0.363). There were significant differences in the contents of IgE of BALF for the asthmatic, DXM and control groups [(23.85 ± 5.97) g/L, (13.15 ± 2.22) g/L, (6.54 ± 1.03) g/L, F = 38.558, P = 0.000]. The percentages of CD8(+)CD28(-)T cell in peripheral blood in asthmatic and DXM groups [(18.68 ± 4.12)% and (13.43 ± 2.91)%] were significantly higher than those in control mice [(8.43 ± 4.60)%, both P0.05]. The percentages of CD8(+)CD28(-)T cell of BALF in asthmatic group and DXM group [(1.25 ± 0.40)% and (0.66 ± 0.49)%] were also significantly higher than those in control mice [(0.21 ± 0.19)%, both P0.05]. The percentages of CD8(+)CD28(-)T cell of blood and BALF in the DXM mice were significantly lower than those in asthmatic group. The correlations between IgE (r = 0.864, P = 0.012), EOS (r = 0.804, P = 0.029) and CD8(+)CD28(-)T cell were significant.The fraction of CD8(+)CD28(-)T cell is closely correlated with the inflammation of asthmatic airway. The airway hyperresponsiveness and inflammation in asthmatic mice may be relieved by DXM through its effect of inhibiting the expression of CD8(+)CD28(-) T cell.

Human lecithin:cholesterol acyltransferase (LCAT, E.C.2.3.1.43) is a serine-type esterase that contains six cysteines, two of which, Cys31 and Cys184, are free. The remaining cysteines form disulfide links. One of these is between Cys50 and Cys74 and the other is between Cys313 and Cys356. The cDNA of LCAT and mutants in which one or two of the six cysteines were replaced by glycine was expressed in COS-6 cells. Polymerase chain reactions and Northern blot analysis indicated that LCAT mRNA was produced by all transfectants. Western blots of all transfected cells probed with a polyclonal antibody revealed intracellular LCAT. Substitution of glycine for either Cys50, Cys74, Cys313, or Cys356 was associated with a nearly total absence of activity in the medium. No protein was secreted when glycine replaced either of the amino acid residues that link Cys313 and Cys356. The small amounts of the Cys50-->Gly and Cys74-->Gly mutants found in the medium had specific activities that were much lower than that of the wild-type LCAT. All other transfectants secreted immunologically measurable amounts of active enzyme. Mutants in which one or both free cysteines, Cys31 and Cys184, were replaced with glycine were less active than the wild type and only partially inhibited by a sulfhydryl blocking reagent. The substrate specificities of the Cys31-->Gly and Cys184-->Gly mutants differed from that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Human plasma phospholipid transfer protein (PLTP) contains six potential N-glycosylation sites (Asn-X-Ser). To study the role of these sites on PLTP structure and function, seven variants in which asparagine (N) residues were converted to glycine (G) were prepared by site-directed mutagenesis. These were N(47)G, N(77)G, N(100)G, N(126)G, N(228)G, N(381)G and N(47, 77, 100, 126, 228, 381)G (N(null)G). These variants and wild-type (WT) PLTP were expressed in COS-7 cells. Intracellular and secreted PLTP mass was analyzed by Western blots and quantitative enzyme-linked immunosorbent assay; PLTP activities in cellular lysates and media were based on the transfer of [(3)H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N(null)G was not detected intracellularly. N(381)G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N(47)G, N(77)G, N(100)G, N(126)G, N(228)G and N(381)G were similar in cell lysate (range = 67-90% WT) and medium (range = 65-77% WT). Intracellular masses of these PLTP variants were similar to that of WT (Mean = 103% WT); mean secreted mass was 88% WT. These results suggest that secretion-competent PLTP requires glycosylation but that no single glycosylation site is required.

To establish a method for rapid molecular detection of Streptococcus pneumoniae and Haemophilus influenzae in the early stage of infection.Specific DNA probes for Streptococcus pneumoniae and Haemophilus influenzae and 16 S rRNA universal probe of bacteria were synthesized by polymerase chain reaction (PCR) and labeled with biotin. The DNA of the bacteria, virus and fungi were hybridized with these probes respectively prior to application for examination of the clinical samples.The DNA probes of 263, 351, and 370 bp were amplified by PCR. Streptococcus pneumoniae and Haemophilus influenzae reacted only with their corresponding probes, and no cross reaction of the bacterial universal probe with virus and fungi was noted. The method could detect bacterial DNA in as small amount as 1 ng. Of the 100 sputum specimens, 11 were found to be positive for Streptococcus pneumoniae and 8 for Haemophilus influenzae, with a positivity rate greater than that by sputum culture.DNA probe detection is simple, rapid, and specific for clinical examination of Streptococcus pneumoniae and Haemophilus influenzae.

To establish a rapid method for detecting Pseudomonas aeruginosa at the early stage of infection.Specific primers were designed according to oprI gene sequence of Pseudomonas aeruginosa, and the specific probe was synthesized by PCR. After photosensitive biotin labeling of the bacterial DNA, reverse dot-blot hybridization was used to detect Pseudomonas aeruginosa.The probe synthesized was highly specific to Pseudomonas aeruginosa without cross reaction with other bacteria, viruses or fungi. The method was capable of detecting 100 ng bacteria DNA.Reverse dot-blot hybridization possesses the merits of speediness and specificity in the detection of Pseudomonas aeruginosa in the early stage of infection.

Diminished apoptosis, a critical event in tumorigenesis, is linked to down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer cells. 13- S -hydroxyoctadecadienoic acid (13- S -HODE), which is the primary product of 15-LOX-1 metabolism of linoleic acid, restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for linoleic and arachidonic acid metabolites. PPAR-δ promotes colonic tumorigenesis. NSAIDs suppress PPAR-δ activity in colon cancer cells. The mechanistic relationship between 15-LOX-1 and PPAR-δ was previously unknown. Our current study shows that ( i ) 13- S -HODE binds to PPAR-δ, decreases PPAR-δ activation, and down-regulates PPAR-δ expression in colorectal cancer cells; ( ii ) the induction of 15-LOX-1 expression is a critical step in NSAID down-regulation of PPAR-δ and the resultant induction of apoptosis; and ( iii ) PPAR-δ is an important signaling receptor for 13- S -HODE-induced apoptosis. The in vivo relevance of these mechanistic findings was demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our findings indicate that the down-regulation of PPAR-δ by 15-LOX-1 through 13- S -HODE is an apoptotic signaling pathway that is activated by NSAIDs.

Human plasma phospholipid transfer protein (PLTP) exchanges phospholipids between lipoproteins and remodels high-density lipoproteins (HDLs). We determined phospholipid transfer activity and HDL binding ability in wild-type PLTP and in 16 PLTP variants created by replacing 12 charged amino acids by site-directed mutagenesis. The data were analyzed in relation to the structure of a member of the same gene family, bactericidal/permeability-increasing protein, which is a boomerang-shaped molecule containing two symmetrical, hydrophobic pockets that bind phospholipid molecules. When expressed in COS-7 cells, wild-type and all mutant PLTPs accumulated intracellularly to nearly the same extent. Relative to wild-type PLTP, substitution(s) for amino acids with a lateral position totally exposed to the solvent produced reductions in transfer activity proportional to the reductions in the level of HDL binding. Variants containing substitutions for charged amino acids on the concave surface of PLTP did not affect binding to HDL or specific transfer activity. A mutation in the C-terminal pocket (E270R) led to a decrease in both the specific transfer activity and the level of binding to HDLs, whereas mutations in the N-terminal pocket (R25E and D231R) resulted in a large decrease in specific transfer activity without affecting HDL binding. The data support a model of transfer in which N- and C-terminal pockets have different roles in HDL binding and transfer activity. The N-terminal pocket may be critical to PLTP transfer activity but may have no involvement in binding to lipoproteins, whereas amino acid substitutions in the C-terminal pocket might reduce PLTP activity by decreasing PLTP's affinity for HDLs.

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys135 and Cys175; these residues, which correspond to Cys129 and Cys168 in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys129 → Gly and PLTP Cys168 → Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys5 → Gly and PLTP Cys318 → Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys5 → Gly and PLTP Cys318 → Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.

AIM: To evaluate the efficacy of furazolidone-based triple and quadruple therapy in eradicating Helicobacter pylori (H. pylori) in a multi-center randomized controlled trial. METHODS: A total of 720 H. pylori positive patients with duodenal ulcer disease were enrolled at 10 different hospitals in Jiangxi province in China. The patients were randomly assigned to four treatment groups as follows: patients in Groups 1 and 3 received rabeprazole (10 mg), amoxicillin (1000 mg) and furazolidone (100 mg) twice daily for 7 and 10 d, respectively; patients in Groups 2 and 4 received rabeprazole (10 mg), bismuth (220 mg), amoxicillin (1000 mg) and furazolidone (100 mg) twice daily for 7 and 10 d, respectively. The primary outcome measure was H. pylori eradication rate 4 wk after treatment by intention-to-treat and per protocol analysis, while the secondary outcome measures were symptom and sign changes at the end of treatment and 4 wk after the end of treatment, as well as the proportion of patients who developed adverse events. RESULTS: The demographic data of the four groups were not significantly different. Overall, 666 patients completed the scheme and were re-assessed with the 13C-urea breath test. The intention-to-treat analysis of the H. pylori eradication rates in Groups 1, 2, 3 and 4 were 74.44%, 82.78%, 78.89% and 86.11%, respectively. The H. pylori eradication rate in Group 4 was significantly higher than that in Group 1. According to the per protocol analysis, the H. pylori eradication rates in Groups 1, 2, 3 and 4 were 81.21%, 89.22%, 85.54% and 92.26%, respectively. The H. pylori eradication rate in Group 4 was significantly higher than that in Group 1. The number of adverse events was 15 (8.3%), 16 (8.9%), 15 (8.3%) and 17 (9.4%) in Groups 1, 2, 3 and 4, respectively, including dizziness, vomiting, diarrhea, nausea, skin rash, itchy skin, and malaise. The symptoms were relieved without special treatment in all of the patients. CONCLUSION: Both 7- and 10-d quadruple furazolidone-based therapies achieve satisfactory H. pylori eradication rates.

Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19--Ala), LCAT (Ser181--Gly), LCAT (Ser208--Ala), LCAT (SEr216--Ala), LCAT (Ser225--Ala) and LCAT (Ser383--Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19--Ala, Asn20--Thr) and LCAT (Ser383--Ala, Asn384--Thr), in order to ascertain the effect of the serines on N-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34-138%). Except for LCAT (Ser181--Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean 95% activity of wild type; range, 65-169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.

There are four potential N-glycosylation site (Asn-X-Ser/Thr) in human lecithin:cholesterol acyltransferase (LCAT, residues 20, 84, 272, and 384). To study the role of the N-linked sugars, the codon for Asn at these positions was replaced with one for Thr (AAC to ACC). The wild-type and mutant LCAT cDNAs were used to transfect COS-6 cells from which RNA was isolated; cDNAs were synthesized by reverse transcription and subjected to the polymerase chain reaction, which showed that all transfectants synthesized LCAT-specific mRNA. No intracellular or secreted LCAT was detected with the Asn272-->Thr transfectants, indicating that this residue is essential for intracellular processing. All other single-point transfectants were secretion-competent. Although there was detectable LCAT protein inside the cells and in the media of the transfectant, Asn84-->Thr, its specific activity and secreted amount were only 26% and 58% of the wild type, respectively. This implies that Asn84 is critical for full activity but not for intracellular processing. The amount secreted, specific activity, and Vmax of LCAT (Asn20-->Thr) were similar to those of the wild-type LCAT. LCAT (Asn384-->Thr) differed from the wild-type LCAT only by a lower Km. These results suggest that glycosylation at residues 20 and 384 is not essential for intracellular processing, secretion, or activity.

Abstract The genetic and biochemical basis of fish-eye disease (FED) was investigated in a 63-year-old female proband with low plasma HDL cholesterol. Analyses of corneal and plasma lipids of the proband were consistent with impaired lecithin:cholesterol acyltransferase (LCAT) activity. Free cholesterol and phospholipid levels were elevated relative to control values, whereas cholesteryl ester levels were greatly reduced. Fatty acid compositions of corneal lipids from the proband and control subjects differ from the respective fatty acid compositions of their plasma lipids. This suggests that the metabolic pathways and acyl chain specificities for phospholipid, cholesteryl ester, and triglyceride metabolism within the cornea are distinct from those of plasma. Sequencing of the LCAT gene from the proband revealed a novel mutation at nucleotide 399, corresponding to an Arg 99 →Cys substitution. Secretion of LCAT (Arg 99 →Cys) by transfected COS-6 cells was ≈50% of that of the wild type, but its specific activity against reassembled HDL was 93% lower than that of wild-type LCAT. The specific activities of wild-type and LCAT (Arg 99 →Cys) against LDL were reduced similarly, suggesting that the appearance of the FED phenotype does not require enhanced activity against LDL. Our data support the hypothesis that FED is a partial LCAT deficiency in which poor esterification in specific types of HDL particles may contribute to the appearance of the corneal opacities.