BACKGROUNDTo express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.

METHODSBovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.

RESULTSCompared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.

CONCLUSIONSThe EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.

Animals ; Catalytic Domain ; genetics ; Cattle ; Cloning, Organism ; DNA, Complementary ; Enteropeptidase ; analysis ; genetics ; Recombinant Proteins

AIMTo detect the residual clenbuterol in pork meat and liver using HPLC with Coulometric electrode array system.

METHODSHomogenized meat or liver sample was treated with 1 mol x L(-1) hydrochloric acid and centrifuged, the fat existing in meat or liver tissue was removed by diethyl ether. The pH of the remaining aqueous layer was adjusted to 10.8 +/- 0.2 or 11.6 +/- 0.2 for meat or liver and liquid-liquid extraction with diethyl ether was followed. The ether extract was evaporated to dryness, the residue was dissolved in the mobile phase. The mobile phase A consisted of 50 mmol x L(-1) phosphoric acid-30 mmol x L(-1) triethylamine and was adjusted to pH 4.0 with 2 mol x L(-1) sodium hydroxide solution. The mobile phase B consisted of methanol-acetonitrile (30:45). A mixture of mobile phase A and B (80:20) was used in the method. A four electrode array module was selected for quantitation, the electrode potentials were set at 450, 600, 650 and 680 mV respectively.

RESULTSThe two calibration curves for meat and liver showed good linearity between 1.88 - 60.16 ng x g(-1), the detection limit of clenbuterol was 1.2 ng x g(-1).

CONCLUSIONThis method using HPLC-electrochemical detection is reproducible, and the sensitivity is good enough for the determination of clenbuterol in meat and liver.

Animal Feed ; analysis ; Animals ; Chromatography, High Pressure Liquid ; methods ; Clenbuterol ; analysis ; Drug Residues ; analysis ; Electrochemistry ; methods ; Electrodes ; Liver ; chemistry ; Meat ; analysis ; Swine

Objective:To observe the outcome after sacral canal injection in patients with disc herniation associated with without sciatica. Methods:From December 2010 to June 2011,65 patients with acute low back pain without sciatica due to lumbar disc herniation or bulging confirmed by CT or MRI were randomly divided into sacral canal injection group (experi-mental group) and lumbar oblique wrench group (control group):the experimental group had 35 cases,including 30 males and 5 females,with an average age of (43.90±1.14) years old ranging from 33 to 56 years old. The control group had 30 cases,in-cluding 27 males and 3 females,with an average age of (44.00±1.19) years old ranging from 34 to 57 years old. The course of morbidity was 1 to 3 days. All patients received sacral canal injection or lumbar oblique wrench method. The visual analog scale (VAS) scores before and at 30 min after treatment were compared between two groups. Results:The symptom of acute low back pain were relieved obviously. The average VAS scores before and after treatment in experimental group were de-creased from 6.63 ±0.97 to 3.06 ±1.51,in control group were from 6.67 ±0.96 to 3.93 ±1.20 respectively. These two methods could improve the VAS score,but the effect of sacral canal injection group was better than that of lumbar oblique wrench group,there was statistically differences (P<0.05). Conclusion:It is effective that the methods of sacral canal injection and lumbar oblique wrench applied to patients with acute low back pain without sciatica due to lumbar disc herniation or bulging confirmed,the former has better effect.

Objective To explore the incidence and risk factors of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgeries and its correlation with preoperative risk assessment of cerebrovascular events,so as to guide perioperative risk management.Methods A retrospective study was conducted on 40 patients aged≥18 years who underwent non-cardiac and non-neurosurgical surgeries and experienced perioperative ischemic stroke in the First Affiliated Hospital of Henan University of Science and Technology from January 2015 to January 2022,forming the stroke group.A control group of 160 patients without perioperative ischemic stroke was selected in a 1:4 case-control ratio,matched for gender,age,date of operation,and the surgeon.Clinical data and preoperative risk assessment of cerebrovascular events(including the single or combined application of head CT/MRI,transcranial Doppler ultrasound,carotid ultrasound,and neurological consultation)of the two groups of patients were collected and statistically analyzed.Multiple logistic regression analysis was used to identify risk factors associated with perioperative ischemic stroke.Results The incidence of perioperative ischemic stroke was 0.042%.Multiple logistic analysis results showed that hypertension(OR=7.858,95%CI 2.175-28.388,P=0.002),hyperlipidemia(OR=4.457,95%CI 1.320-15.049,P=0.016),renal insufficiency(OR=8.277,95%CI 1.480-46.282,P=0.016),and intraoperative hypotension(OR=3.862,95%CI 1.211-12.317,P=0.022)were independent risk factors for perioperative ischemic stroke in non-cardiac and non-neurological surgeries;preoperative cerebrovascular risk assessment(OR=0.130,95%CI 0.031-0.542,P=0.005)was a protective factor against it.Conclusions The incidence of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgery is low but has a poor prognosis.Hypertension,hyperlipidemia,renal insufficiency,and postoperative hypotension are risk factors for perioperative ischemic stroke,while preoperative cerebrovascular event risk assessment is beneficial to reducing its incidence.

Air Pollution ; Asthma/drug therapy* ; China ; Humans ; Treatment Adherence and Compliance