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BACKGROUND: Aspergillus oryzae protease can release the opioid peptide ß-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. RESULTS: A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with µ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. CONCLUSION: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.
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Stroke rescue features strong time sensitivity and high complexity. Minimizing the time of consumption in pre-hospital and in-hospital stroke rescue is key to improve stroke rescue efficiency and reduce the disability rate. In December 2017, a tertiary hospital launched the construction of a one-stop stroke rescue platform. This platform was centered on " multi-mode image fusion operating room" , operating as a one-stop rescue mode integrating emergency admission, imaging examination, intravenous thrombolytic therapy, mechanical thrombolytic therapy, postoperative evaluation, and so on. The seamless convergence workflow of pre-hospital, in-hospital and post-hospital could effectively optimize the physical rescue pathway. In order to ensure the efficient and orderly operation of the platform, the hospital adopted such measures as multidisciplinary integration, pre-hospital and in-hospital integration construction, and regional stroke care network. Since its operation in September 2019, the platform has treated more than 1 000 patients by December 2021. The application of the platform had effectively improved the efficiency of stroke rescue, led the development of regional stroke rescue system, and provided the reference for raising the stroke rescue capacity and management level in China.
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Objective:To investigate the difference of metabolomics between acute heart failure (AHF) patients and control. To find and validate new metabolic biomarkers.Methods:This was a single-center case-control study which included 89 acute heart failure patients admitted to the emergency department of Henan Provincial People's Hospital from January 2018 to June 2019. Eighty people without heart failure and diastolic dysfunction were enrolled as control group whose age and sex were matched to the study group. The fasting blood samples were collected from femoral arterial. Qualitative and quantitative analyses of plasma metabolites were performed in 2 groups by high performance liquid chromatography tandem mass spectrometry (UHPLC-MS), Orthogonal partial least squares-discriminant analysis (OPLS-DA) model and ROC curve method were applied.Results:Compared with the control group, we found that AHF group had higher likelihood to groups with coronary heart disease (37% vs. 7%, P<0.001), hypertension (58% vs. 28%, P<0.001), diabetes (33% vs. 18%, P=0.033), atrial fibrillation (24% vs. 4%, P<0.001), smoking history (42% vs. 18%, P=0.001), and that AHF group had higher creatinine level [(121.6 ± 78.4) vs. (69.0 ± 21.0), P<0.001], higher urea level [(11.5 ± 7.6) vs. (6.2 ± 2.0), P<0.001], higher heart rate [(92 ± 23) vs. (78 ± 14), P<0.001], hypoproteinemia [(32.4 ± 5 .2) vs. (40.4 ± 2.2), P<0.001], and significantly increased BNP level [(4 200 ± 5 229) vs. (100 ± 68), P<0.001], lower left ventricular ejection fraction[(45 ± 8) vs. (57 ± 6), P<0.001], low serum sodium level ( P<0.001). The metabolites of AHF group were significantly different from those of the control group. The metabolites involved amino acids, fatty acids, lipids, nucleosides and their derivatives. Adenine, N-acetyl-L-glutamic, pseudouridine and Gamma-Glutamylcysteine had certain diagnostic value for AHF comparing to control. The AUC were 0.995, 0.932, 0.920 and 0.900. And the AUC value for BNP diagnosis of AHF is 0.978. Conclusions:There were significant differences in metabolism between AHF group and control group including multiple substances. Adenine, N-acetyl-L-glutamic, pseudouridine and Gamma-Glutamylcysteine has similar diagnostic value compared with BNP for diagnosing AHF.
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Objective:To discuss the relationship between neurofunctional prognosis and early mean arterial pressure (MAP) of patients with post-cardiac arrest syndrome (PCAS).Methods:This retrospective study enrolled 151 PCAS patients in Henan Provincial People’s Hospital between January 2016 and January 2018. A multivariate logistic regression analysis was established to determine the correlation between the MAP at the first 6 h and the prognosis of the patients at discharge. The effect of angioactive drugs on the neurofunctional prognosis of patients at discharge was determined by chi-square test.Results:The multivariate logistic regression analysis showed that the TWA-MAP of patients with good neurofunctional prognosis at discharge was significantly higher than that of patients with poor prognosis (83.25±13.69) mmHg vs (77.06±18.37) mmHg, P=0.042. In the first 6 h after ROSC, the neurofunctional prognosis of patients with good prognosis in the TWA-MAP value of 71 to 80 mmHg was significantly better than that of patients with TWA-MAP lower than 70 mmHg (11% vs 37%, P=0.009). The variable logistic regression analysis results showed that the level of TWA-MAP higher than 70 mmHg or 65 mmHg was the independent factor affecting the outcome, while the level of TWA-MAP higher than 70 mmHg had a better correlation with the neurofunctional prognosis ( OR=4.11, 95% CI:1.34-12.66, P=0.014). In patients with TWA-MAP higher than 70 mmHg, the neurofunctional prognosis of patients with good prognosis but without angioactive drugs was significantly better than that of those with angioactive drugs (48% vs 24%, P=0.010). Conclusion:Holding TWA-MAP higher than 70 mmHg in the first 6 h after PCAS is helpful to improve the neurofunctional prognosis of patients at discharge.
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Objective@#To investigate the effects of apolipoprotein E deficiency (Apo E-/-) on plasma and lipoprotein distribution of sphingosine-1-phosphate (S1P) in mice.@*Methods@#Five male or female Apo E-/- or wild type (WT) mice were fed with chow diet and sacrificed at 32-week-age and plasma was collected. The constituents of lipoprotein(very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL)) were separated by ultracentrifuge. The protein concentration of constituents was detected by BCA protein quantitative kit, and the S1P concentration in plasma and various lipoprotein constituents was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blot was used to determine the plasma, liver, and kidney protein expression of apolipoprotein M(Apo M), which is considered as specific ligand of S1P.The S1P concentration in plasma and various constituents of lipoprotein in the Apo E-/- mice was compared to respective WT mice.@*Results@#(1)Plasma S1P content was significantly higher in the Apo E-/- groups than that of WT groups (male: (535.7±78.5)nmol/L vs. (263.3±22.0)nmol/L; female: (601.1±64.0)nmol/L vs. (279.0±33.9)nmol/L; all P<0.01). (2) Compared with WT mice, S1P content in non-HDL(LDL+ VLDL) was significantly higher in Apo E-/- mice (male: (504.9±52.8)nmol/L vs. (28.7±9.0)nmol/L; female: (427.7±27.4) vs. (27.8±4.7)nmol/L; after standardization of protein concentration, male: (385.0±41.2)pmol/mg protein vs. (71.4±6.6)pmol/mg protein; female: (330.2±22.0)pmol/mg protein vs. (67.2±12.1)pmol/mg protein; all P<0.01). (3) The expression of Apo M in plasma, liver and kidney was significantly higher in Apo E-/- groups than that of WT groups(all P<0.05).@*Conclusion@#The deficiency of Apo E could lead to upregulated S1P expression in the non-HDL, the underlying mechanism might be the increased transfer of HDL into the non-HDL by Apo M-S1P.
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[ABSTRACT]AIM:Toinvestigatetheinteractionandthemechanismofsphingosine-1-phosphate(S1P)and phospholipid transfer protein (PLTP) in lipoprotein.METHODS:The S1P content in the plasma and lipoprotein from 10-week-old PLTP transgenic (PLTP-Tg) mice and wild-type (WT) mice (n=8 each) was assayed.The transport of S1P by PLTP was determined by S1P transfer assay.The content of specific S1P carrier, apolipoprotein M, was detected by West-ern blotting.RESULTS:Plasma S1P contents were decreased by 21.1%in PLTP-Tg mice compared with WT mice.S1P content in high-density lipoprotein ( HDL) fraction ( HDL-S1P) from PLTP-Tg mice was decreased by 35.1% compared with WT mice, whereas the S1P in low-density lipoprotein (LDL) fraction (LDL-S1P) was increased by 127.4%.The re-sults of S1P transfer assay indicated that PLTP facilitated S 1P transport from erythrocyte to recombinant liposome at 37℃in D-Hanks buffer solution .The plasma content of apolipoprotein M was not changed in PLTP-Tg mice compared with WT mice.CONCLUSION:PLTP is a key factor to maintain plasma HDL-S1P under physical condition .Overexpression of PLTP decreases the HDL-S1P but increases LDL-S1P.The mechanism might be related to the capability of PLTP on trans-ferring S1P from erythrocyte to lipoprotein.
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AIM:To establish a liquid chromatography method for determining the monosaccharide composition of human lipoproteins, and to investigate the differences between diabetic patients and healthy participants.METHODS:Liquid chromatography with pre-column derivatization was used to determine the neutral and basic monosaccharides, and liquid chromatography tandem mass spectrometry was applied to quantify N-acetylneuraminic acid content.RESULTS:The contents of mannose, glucosamine, N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in high-density lipoprotein from healthy participants and diabetic patients were (5.88 ±0.94),(16.49 ±4.11),(1.31 ±0.33), (0.87 ±0.16), (7.18 ±1.64), (2.14 ±0.12) mmol/(g protein) and (8.68 ±0.39), (24.73 ±5.50), (1.91 ±0.54), (1.23 ±0.35), (9.73 ±2.85), (3.53 ±0.27) mmol/(g protein), respectively.The contents of mannose, glu-cosamine,N-acetylglucosamine, glucose, galactose and N-acetylneuraminic acid in low-density lipoprotein from healthy par-ticipants and diabetic patients were ( 29.20 ±3.57 ) , ( 50.77 ±4.72 ) , ( 5.28 ±0.64 ) , ( 10.06 ±1.37 ) , ( 28.44 ± 3.96),(6.86 ±0.11) mmol/(g protein) and (30.08 ±3.78), (38.52 ±6.38), (3.79 ±0.78), (7.63 ±1.50), (20.05 ±2.63), (6.45 ±0.18) mmol/(g protein), respectively.The contents of mannose, glucosamine, glucose, ga-lactose and N-acetylneuraminic acid in very-low-density lipoprotein from healthy participants and diabetic patients were (91.21 ±4.12), (27.05 ±2.34), (4 230.95 ±15.83), (43.40 ±3.75), (2.95 ±0.24) mmol/(g protein) and (82.40 ±0.51), (30.16 ±0.32), (4 722.73 ±93.27), (34.05 ±2.81), (4.42 ±0.15) mmol/(g protein), respec-tively.CONCLUSION:Liquid chromatography with pre-column derivatization is suitable for the neutral and basic mono-saccharide analysis in human lipoproteins, and the glycosylation of lipoproteins in diabetic patients are significantly changed compared with the healthy controls.
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A simple and sensitive capillary electrophoresis method with field-enhanced stacking concentration for the analysis of protocatechuic aldehyde, protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B in Salvia miltiorrhiza var. miltiorrhiza f. alba was developed. The separation was achieved with a fused-silica capillary (75 microm x 50.2 cm, effective length was 40 cm) and a running buffer 15 mmol x L(-1) borax (pH 10.0) containing 20% CH3 OH. The UV detection wavelength was 210 nm. The applied voltage was 28 kV, and the cartridge temperature was 25 degrees C. Water plug was introduced from the anode by 0.5 psi x 4 s before injection. Sample was injected by electrokinetic injection - 8 kV x 3 s. The linear range of protocatechuic aldehyde is 3.0-60.00 mg x L(-1) (R2 = 0.999 8); that of protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B are 1.0-20.00 mg x L(-1) (R2 are 0.999 1, 0.999 4, 0.998 9 and 0.999 8, respectively), and the limits of detection of five analyts are 0.55, 0.40, 0.25, 0.32, 0.38 microg x L(-1), respectively, Stacking factor is higher and precision is satisfactory. The recoveries ranges were from 97.3% to 99.8%. The proposed method was used to determine the protocatechuic aldehyde, protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B in S. miltiorrhiza var. miltiorrhiza f. alba. The proposed method is simple, rapid, accurate and high sensitivity, and can be used to control of the quality of S. miltiorrhiza var. miltiorrhiza f. alba.
Subject(s)
Benzaldehydes , Benzofurans , Catechols , Cinnamates , Depsides , Electrophoresis, Capillary , Methods , Hydroxybenzoates , Lactates , Plant Extracts , Quality Control , Salvia miltiorrhiza , Chemistry , Sensitivity and Specificity , Solubility , WaterABSTRACT
OBJECTIVE:To establish a high performenc capillary zone electrophoresis method for the determination of the water-soluble active ingredients in different parts of the Salvia miltiorrhiza.METHODS:Capillary electrophoresis was conducted using uncoated capillary column(75 ?m?50.2 cm,effective length=40 cm)with 5 mmol?L-1 phosphate-borax(ph7.4)as running buffer by pressure injection(0.5 psi/4 s)and constant voltage separation(20 kV)with a detection length of 210 nm and a column temperature of 25 ℃.RESULTS:A complete baseline separation of PAH,DSS and PA was achieved within 8 min under the optimized conditions.The good linear range was from 2.5 ?g?mL-1 to 200.0 ?g?mL-1 for all the three ingredients.The average recoveries of the 3 ingredients were 100.04%,99.99%,and 100.01%,respectively,with RSD at 1.75%,1.73%,and 1.74%,respectively.CONCLUSION:The method is satisfactory in precision,recovery and linearity,and it can be used to determine three water-soluble active ingredients in different parts of the Salvia miltiorrhiza.
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Objective To find an ideal method of DNA isolation from blood and especially from clotted blood and to minimize the volume of blood collected for laboratory and clinical tests.Methods DNAs were isolated from antiagglutinated and agglutinated blood samples from auricular veins of 30 healthy subjects. The DNAs of these samples were obtained by a nonenzymatic, nontoxic procedure optimized by us and determinated by agarose gel electrophoesis and PCR. Results The yields of DNA isolated from clotted blood and antiagglutinated blood were (40.2?8.86)mg DNA/L and (39.1?10.2)mg DNA/L, and purities were 1.87?0.11 and 1.92? 0.12. The DNAs that we isolated from all samples had high molecular weight and by PCR the dimorphism of ALU alleles of the 8th intron of t-PA was easy to be obtained, so they were complete and reliable. Conclusion This method is rapid, easy, efficient and nontoxic for isolation of DNA from clotted and fresh blood and meets requirements for clinical testing and molecular biology study.