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1.
Chinese Journal of Biotechnology ; (12): 2126-2140, 2023.
Article in Chinese | WPRIM | ID: wpr-981194

ABSTRACT

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.


Subject(s)
Transaminases/chemistry , Molecular Docking Simulation , Amines/chemistry , Pyruvic Acid/metabolism , Enzyme Stability
2.
Article in Chinese | WPRIM | ID: wpr-991127

ABSTRACT

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca2+,reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca2+overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

3.
Article in Chinese | WPRIM | ID: wpr-1022795

ABSTRACT

Objective:To compare the efficacy and safety of low-intensity illumination 3D heads-up system-assisted pars plana vitrectomy (PPV) and traditional microscope eyepiece system-assisted PPV for proliferative diabetic retinopathy (PDR).Methods:A randomized controlled study was conducted.Forty patients (40 eyes) who were diagnosed as PDR and met the PPV standard were included in Xuzhou First People's Hospital from June to December 2022.The patients were randomly divided into 3D group and eyepiece group using a random number table method, with 20 eyes in each group.The eyes in 3D group underwent 3D heads-up system-assisted PPV, and the eyes in eyepiece group received traditional microscope eyepiece system-assisted PPV.The intravitreal injection of 0.5 mg(0.05 ml) ranibizumab was performed 6 or 7 days prior to three-channel 25G PPV for all the eyes.The brightness of endoilluminator was adjusted to minimum level during the surgical procedure, and the brightness of the optical fiber and chandelier in 3D group was set to 20%, while that in eyepiece group was 32% and 46%, respectively, and was further matched to the actual requirements of the surgery.The light intensity of optical fiber and chandelier was measured at 5 mm and 10 mm with a digital photometer.Best corrected visual acuity (BCVA) was measured before surgery and 7 days, 1 month and 3 months after surgery.Electroretinogram (ERG) was recorded by the Retiscan before surgery and 1 month after surgery to evaluate retinal function.Intraocular pressure and postoperative complications in both groups were compared.This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of The Affiliated Xuzhou Municipal Hospital of Xuzhou Medical University (No.xyy11[2022]027). Written informed consent was obtained from each subject prior to entering the cohort.Results:The BCVA was 2.21±1.13, 1.99±1.07, 1.26±0.86 and 0.98±0.65 in 3D group, and 1.89±0.95, 1.94±0.79, 1.42±0.80 and 1.31±0.79 in eyepiece group at before surgery and 7 days, 1 month, and 3 months after surgery, respectively.There was no significantly intergroup difference in BCVA ( Fgroup=0.022, P=0.884). The BCVA was significantly different at various time points before and after surgery ( Ftime=18.765, P<0.001). The BCVA was significantly improved at 1 and 3 months after surgery in 3D group and at 3 months after surgery in eyepiece group in comparison with before surgery, showing statistically significant differences (all at P<0.05). There were significant differences in the latency of dark-adapted 3.0 a-wave before and after surgery between two groups ( Htime=3.983, P=0.046), and the latency of dark-adapted 3.0 a-wave was shorter after surgery than before surgery in both groups (all at P<0.05). The light intensities of optical fiber and chandelier at 5 mm and 10 mm during surgery were lower in 3D group than in eyepiece group, and the differences were statistically significant (all at P<0.001). There was no significant difference in intraocular pressure between the two groups at different time points ( Fgroup=0.980, P=0.328; Ftime=2.706, P=0.062). There was no significant difference in the number of postoperative vitreous hemorrhage between the two groups ( χ2=0.960, P=0.327). Conclusions:Low-intensity illumination 3D heads-up system-assisted PPV has the same outcome as traditional microscope eyepiece system-assisted PPV for PDR.However, compared with the traditional microscope eyepiece system, the light intensity on the retina from low-intensity illumination 3D heads-up system is lower on the retina during surgery and therefore produce less light damage to retinal function of patients.

4.
Journal of Pharmaceutical Practice ; (6): 136-139,146, 2018.
Article in Chinese | WPRIM | ID: wpr-790851

ABSTRACT

Objective To compare the renal toxicity of vancomycin with continuous infusion vs intermittent infusion. Methods The databases of EMBASE,PUBMED,the Cochrane Register of Controlled Trials,CBM,CNKI and WanFang were searched.The Cochrane Revman5.2 software was used for Meta-analysis.Results Two RCTs and eight observational studies were included in the systematic literature search with total of 1 764 patients.1 037 patients received vancomycin with continuous infusion while 727 patients with intermittent infusion.The Meta-analysis indicated that there was no significant difference in renal toxicity between continuous infusion group and intermittent infusion group(P>0.05).Conclusion Vanco-mycin continuous infusion cannot effectively reduce the incidence of renal toxicity.

5.
Article in Chinese | WPRIM | ID: wpr-808410

ABSTRACT

Objective@#The aim of this study was to investigate the association of both peripheral and central systolic blood pressure (pSBP and cSBP) with urinary albumin-to-creatinine ratio (UACR) in a community-based population in Beijing.@*Methods@#A total of 3 479 Chinese subjects with questionnaire, UACR, pSBP, and cSBP data available were included from an atherosclerosis cohort of Peking University First Hospital in Shijingshan District, Beijing followed up from April to July in 2014. Multivariate linear regression analyses were used to examine the effect of pSBP and cSBP on lnUACR, and further tests for interactions were performed according to associated covariates.@*Results@#Subjects were (59.0±8.6) years old, 36.2% (n=1 260) were male, 46.0% (n=1 595) had hypertension, and 20.2% (n=700) had diabetes. The pSBP and cSBP was (126.9 ± 16.4) mmHg (1 mmHg=0.133 kPa), and (136.3 ± 16.7) mmHg, respectively. P50 (P25-P75) of UACR was 6.2 (4.2-11.1) mg/g. Both pSBP and cSBP were linearly associated with lnUACR adjusted for age, sex, body mass index, smoking status, drinking status, triglyceride, HDL-C, LDL-C, fasting glucose, creatinine, history of cardiovascular disease, antihypertensive and hypoglycemic agents (every 10 mmHg increase for pSBP: β=0.12, 95%CI: 0.10-0.15, P<0.001; for cSBP: β=0.11, 95%CI: 0.09-0.14, P< 0.001). The relationships were remained in subgroups such as non-hypertension group, non-diabetes group, normal UACR group, and 3-combination group (every 10 mmHg increase for pSBP: β=0.09, 95%CI: 0.05-0.13; β=0.12, 95%CI: 0.10-0.15; β=0.07, 95%CI: 0.06-0.09; β=0.08, 95%CI: 0.05-0.12. for cSBP: β=0.07, 95%CI: 0.04-0.11; β=0.11, 95%CI: 0.08-0.13; β=0.07, 95%CI: 0.05-0.08; β=0.06, 95%CI: 0.03-0.09, all P<0.001). Furthermore, analyses for interaction found that both pSBP and cSBP were more strongly associated with lnUACR in males, current smokers and subjects with high serum creatinine level (≥87 µmol/L) when compared with females, non-current smokers and subjects with low serum creatinine level (<87 µmol/L), respectively (all P for interaction<0.05).@*Conclusion@#The results showed that both pSBP and cSBP were independently associated with UACR in this Chinese community-based population even in low risk population suggesting well-controlled both peripheral and central blood pressure may reduce urinary albumin. Males, current smokers and subjects with higher serum creatinine should pay more attention to the impacts of pSBP and cSBP on UACR.

6.
Article in Chinese | WPRIM | ID: wpr-487353

ABSTRACT

Objective To study the effects of QRS-complex duration of patients with chronic left heart failure on their in-hospital prognosis. Methods Total 174 patients admitted for chronic left heart failure ( New York Heart Association class 3 and 4 ) from January 2014 to June 2015 were enrolled the study. They were divided into two groups according to the QRS duration at admission:normal QRS duration group (QRS ≤120 ms, n=145) and prolonged QRS group (QRS ﹥120 ms, n=29). The differences of clinical characteristics and incidences of exacerbated left heart failure, fatal arrhythmias and cardiac death during hospitalization were compared between the two groups. The influences of QRS duration on in-hospital adverse cardiovascular events was analyzed by logistic regression. Resu1ts The proportion of males (75. 9% vs. 24. 1%, P=0. 001), plasma B-type natriuretic peptide (BNP) (7. 1 ± 0. 8 vs. 6. 6 ± 1. 0, P=0. 02), left ventricular end diastolic diameter (LVEDd) [(60. 7 ± 9. 9)mm vs. (53. 5 ± 10. 8)mm, P=0.001], left ventricular end systolic diameter (LVESd) [(49.1 ±13.3)mm vs. (39.7 ±13.0)mm, P﹤0. 001], and the incidence of exacerbated left heart failure (20. 7% vs. 4. 8%, P = 0. 003), fatal arrhythmias (55. 2% vs. 21. 4%, P ﹤0. 001) and cardiac death (6. 9% vs. 0. 7%, P =0. 019) during hospitalization were significantly higher in the prolonged QRS group than in the normal QRS group. Left ventricular ejection fraction( LVEF) in the prolonged QRS group was significantly lower than in the normal QRS group (39. 6% ±17. 3% vs. 50. 5% ± 17. 3%, P =0. 002). Heart rates [(92. 4 ± 21. 4)bpm vs. (81. 6 ± 19. 9)bpm,P=0. 035], plasma BNP(7. 2 ± 0. 8 vs. 6. 7 ± 1. 0, P=0. 029), LVEDd(63. 5 ± 9. 1 vs. 57. 9 ± 9. 1, P=0. 015), LVESd (52. 9 ± 12. 2 vs. 44. 3 ± 11. 8, P=0. 005), incidences of left heart failure deterioration (18. 2% vs. 3. 2%, P=0. 018), fatal arrhythmias (63. 6% vs. 36. 5%, P=0. 027) and cardiac death ( 9. 1% vs. 0%, P=0. 015 ) during hospitalization among male patients in the prolonged QRS group were significantly higher than those in the normal QRS group, but LVEF ( 35. 0% ± 15. 3%vs. 47. 1% ± 16. 2%, P =0. 003 ) was on the opposite. The incidence of left heart failure deterioration among female patients in the prolonged QRS group was higher than that in the normal QRS group ( 28. 6%vs. 6. 1%, P=0. 034). QRS complex duration was positively related to LVEDd ( r=0. 4019, P﹤0. 001) and LVESd ( r =0. 3289, P ﹤0. 001 ) . LVEF in male patients was significantly lower than in female patients (40. 0% ± 16. 7% vs. 53. 2% ± 17. 6%, P﹤0. 001). On the contrary, LVEDd [(59. 4 ± 9. 4) mmvs. (50.3±10.6)mm,P﹤0.001],LVESd[(46.6±12.5)mmvs. (36.2±12.4)mm,P﹤0.001] were greater in male patients than in female patients. After adjusting for gender , age, cigarette smoking, history of high blood pressure, serum creatinine, low-density lipoprotein cholesterol, LVEF, LVEDd, LVESd, use of angiotensin converting enzyme inhibitors ( ACEI) or angiotensin receptor blockers ( ARB) and aldosterone receptor blockers, multiple logistic regression analysis showed that prolonged QRS complex duration is an independent risk factor of adverse prognosis for the patients with HF during hospitalization (OR=4. 21,95%CI:1. 59-11. 12,P=0. 004), and female gender is a protective factor for them ( OR=0. 304,95%CI:0. 116-0. 793,P=0. 015). Conc1usions The incidences of left heart failure deterioration, fatal arrhythmias and cardiac death in the chronic left heart failure patients with prolonged QRS duration were higher than in those with normal duration. Female gender is a protective factor for chronic left heart failure.

7.
Chinese Pharmacological Bulletin ; (12): 970-974, 2016.
Article in Chinese | WPRIM | ID: wpr-495136

ABSTRACT

Aim To study the influence of rosuvastatin on toll-like receptor 4 ( TLR4 ) , and its downstream nu-clear transcription factor NF-kappa B p65, IκBα, in-flammation factors TNF alpha in rats with myocardial hypertrophy induced by pressure overload .Methods Myocardial hypertrophy was induced by abdominal aor-tic constriction ( AAC ) .Male Wistar rats were divided into 3 groups(n=10):① sham-operated rats(S);②AAC rats(M);③AAC+rosuvastatin(10 mg· kg -1· d-1 ) rats.From 1 week pre-opertion to 4 weeks post-operation, three groups were performed by gavage ad-ministration with equal volume of rosuvastatin and vehi-cle.The rats underwent cardiac color Doppler exami-nation.The level of ANP,TLR4, NF-κB p65,IκBα, TNF-αmRNA and protein expression in myocardium were detected by real-time PCR, Western blot, immu-nohistochemical and ELISA respectively .Results The sizes of heart and cardiomyocytes and the expression of ANP mRNA and TLR4 signaling molecules were signif-icantly increased in group M , which could be blocked by rosuvastatin .TLR4 protein was positively related to ANP, NF-κB p65 and TNF-αrespectively .Conclusion Rosuvastatin prevents cardiac hypertrophy induced by pressure overload , which is associated with its inhi-bition of TLR4, NF-κB and TNF-αin myocardium ex-pression .

8.
Chinese Journal of Biotechnology ; (12): 1753-1763, 2015.
Article in Chinese | WPRIM | ID: wpr-337460

ABSTRACT

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.


Subject(s)
Humans , Antimicrobial Cationic Peptides , Antineoplastic Agents , Metabolism , Escherichia coli , Metabolism , Genetic Vectors , HL-60 Cells , Polymerase Chain Reaction , Recombinant Proteins , Sequence Analysis, DNA
9.
Article in Chinese | WPRIM | ID: wpr-291607

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the correlation between smoking status and carotid plaque in rural population residing in Eastern part of China.</p><p><b>METHODS</b>Between July and September of 2013, an epidemiological survey was carried out in residents aged 40 or above men who were enrolled randomly in rural areas of Anqing, Anhui province and Lianyungang, Jiangsu province. The data on epidemiological characteristics including smoking status, physical examination were collected using standardized protocol, and carotid ultrasonography was applied to examine the incidence of carotid plaque among never smokers, former smokers and current smokers. Logistic regress analysis was performed to determine the effect of smoking on carotid plaque.</p><p><b>RESULTS</b>In the study, a total of 625 male participants were included in the study. 51.4% (321 cases) were current smokers, 21.3% (133 cases) were former smokers, and 27.4% (171 cases) were never smokers. 32.0% (200/625) had carotid plaque. The incidence of carotid plaques was significantly higher in current smokers (35.2%, 113/321) than that in never smokers(23.4%,40/171) (χ(2) = 7.26, P = 0.007) and the incidence in former smokers (35.3%, 47/133) was also higher than that in never smokers (23.4%, 40/171) (χ(2) = 5.23, P = 0.022). Multiple logistic regression analysis showed that current cigarette smoking is significantly associated with the increased risk of carotid plaque (OR = 1.84, 95% CI: 1.13-2.98, P = 0.014) in comparison with never smokers, and there was an interaction between current smoking and age in association with carotid plaque. Compared with the young (≤60 years old) and never smoking group (8%, 3/40), prevalence of carotid plaque among the elderly (>70 years old) and smoking group (55%, 31/56) was significantly higher (OR = 8.06, 95% CI: 2.07-31.45) after adjusting for age, systolic blood pressure, diastolic blood pressure, blood glucose, total cholesterol, triglyceride high-density lipoprotein, body mass index, drinking and regional differences.</p><p><b>CONCLUSION</b>It found that cigarette smoking was associated with increased risk of carotid plaque in rural elderly population residing in Eastern part of China.</p>


Subject(s)
Aged , Humans , Male , Middle Aged , Alcohol Drinking , Blood Glucose , Blood Pressure , Body Mass Index , Carotid Stenosis , China , Cholesterol , Lipoproteins, HDL , Prevalence , Risk Factors , Rural Population , Smoking , Triglycerides
10.
China Pharmacy ; (12)2005.
Article in Chinese | WPRIM | ID: wpr-525966

ABSTRACT

OBJECTIVE:To establish a method for content determination of the total naphthoquinone in oil/water emul?sion of shikonin by ultraviolet spectrophotometry.METHODS:The content of the total naphthoquinone was counted in L-shikonin,methanol was used as solvent and the wavelength was set at516nm.RESULTS:The concentration of L-shikonin had good linear correlation with absorbance in the range of8.32~41.60?g/ml(r=1.0000,n=5),the average recovery was98.6%(RSD=0.53%).CONCLUSIONS:The present method is simple,precise and replicable resulting in high recovery and accuracy,it can be used to determine the content of total naphthoquinone in oil/water emulsion of shikonin.

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