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1.
Biomedical and Environmental Sciences ; (12): 864-874, 2015.
Article in English | WPRIM | ID: wpr-258866

ABSTRACT

<p><b>OBJECTIVE</b>In Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.</p><p><b>METHODS</b>NAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.</p><p><b>RESULTS</b>The IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.</p><p><b>CONCLUSION</b>Feedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.</p>


Subject(s)
Animals , Arginine , Corynebacterium , Genetics , Metabolism , Escherichia coli , Feedback, Physiological , Gene Deletion , Mutagenesis, Site-Directed , Phosphotransferases (Carboxyl Group Acceptor) , Genetics , Proline , Metabolism
2.
Biomedical and Environmental Sciences ; (12): 134-137, 2014.
Article in English | WPRIM | ID: wpr-247072

ABSTRACT

A strip reader based lateral flow immunoassay (LFIA) was established for the rapid and quantitative detection of ractopamine (RAC) in swine urine. The ratio of the optical densities (ODs) of the test line (AT) to that of the control line (AC) was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration (IC50) of 0.59 ± 0.06 ng/mL. The limit of detection (LOD) of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.


Subject(s)
Animals , Immunoassay , Phenethylamines , Urine , Reagent Strips , Swine , Urine
3.
Biomedical and Environmental Sciences ; (12): 436-443, 2014.
Article in English | WPRIM | ID: wpr-270583

ABSTRACT

<p><b>OBJECTIVE</b>Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.</p><p><b>METHODS</b>Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.</p><p><b>RESULTS</b>Arginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.</p><p><b>CONCLUSION</b>The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.</p>


Subject(s)
Arginine , Bacterial Proteins , Chemistry , Genetics , Metabolism , Corynebacterium , Genetics , Metabolism , Gene Expression Regulation, Bacterial , Repressor Proteins , Chemistry , Genetics , Metabolism
4.
Chinese Journal of Integrated Traditional and Western Medicine ; (12): 37-40, 2011.
Article in Chinese | WPRIM | ID: wpr-261028

ABSTRACT

<p><b>OBJECTIVE</b>To explore the impacts of Yishen Jiangzhuo Granule (YJG) on peripheral blood B-cells and regulatory T-cells (Treg) in patients with chronic renal insufficiency (CRI).</p><p><b>METHODS</b>Fifty-three CRI patients were randomly assigned to two groups, the control group and the YJG group. Before and after treatment, the following parameters in blood were detected: the peripheral Treg, percentage (CD19+), activation rate (CD19+ CD69+) and apoptotic rate (AV) of B-lymphocyte by flow cytometry; cytokines (IL-6 and IL-10) by CBA stream protein analyzing system; high sensitivity C-reactive protein (hs-CRP) by scattering turbidimetric analysis; homocysteine (Hcy) by end-point method; hemoglobin (HGB) content by Beckman-Coulter hemo-analyser; blood contents of Ca, phosphate (P), blood urea nitrogen (BUN), creatinine (SCr) and plasma albumin (Alb) by automatic biochemical analyser; and urinary contents of creatinine (UCr) by inverse HPLC. Then the product of calcium-phosphate (Ca x P) was calculated based on blood contents of Ca2 and P and the clearance rate of endogenous creatinine (CCr) was calculated based on blood BUN and SCr.</p><p><b>RESULTS</b>After treatment CD19+ and CCr significantly increased (P < 0.01), but AV and SCr decreased in both groups (P < 0.01), with the changes in the YJG group were more significant than those in the control group (P < 0.05); levels of CD19+ CD69+, Treg, IL-6, IL-10, CRP, BUN, P and Ca x P showed no significant change (P > 0.05); levels of Ca2+, HGB and Alb increased as well as of Hcy in both groups (P < 0.05). Correlation analysis: There were negative correlation in CD19+ with AV and Hcy; Alb with AV and Hcy; CCr with CRP, SCr and BUN, while positive correlation existed in SCr with CRP and BUN; and CRP with BUN.</p><p><b>CONCLUSIONS</b>YJG can improve renal function, and delay the progress of renal failure, and it also shows the regulatory effect on B lymphocytes by lowering the apoptosis rate and improving the percentage of CD19+ in patients.</p>


Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , B-Lymphocytes , Metabolism , Drugs, Chinese Herbal , Therapeutic Uses , Kidney Failure, Chronic , Drug Therapy , Metabolism , Phytotherapy , T-Lymphocytes, Regulatory , Metabolism
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