Objective The study was designed to compare the effects of desflurane and isoflurane on the vecuronium-induced neuromuscular block in the elderly patients. Methods Thirty ASA class I - II elderly patients aged over 70 yr undergoing elective surgery under general anesthesia were randomly divided into 3 groups: desflurane group ( I , n = 10) ; isoflurane group ( II , n = 10) and 3 control group ( III , n = 10). Anesthesia was induced with midazolam 0.02-0.05 mg? kg-1 , propofol 0.5-2.0 mg ? kg-1 and fentanyl 2-5?g? kg-1 maintained with inhalation of 6% desflurane(1 MAC) +50% N2O in oxygen (group I ) or 1.15% isoflurane + 50% N2O in oxygen(group II ) or 50% N2O in oxygen (group III ) supplemented with intermittent iv boluses of propofol and fentanyl when necessary. Neuromuscular block was monitored using accelograph (TOF GUARD , Denmark) .A total dose of vecuronium 40 mg ?kg-1 was divided with 4 equal doses of 10?g ? kg-1 , which was administered accumulatively in each patient. The next dose was given when the effect of the previous dose had reached its peak (T1 was no longer depressed in the height of 3 successive stimuli) .The cumulative dose-response curves of the 3 groups were established. The onset time and maximum depression of T1 of the initial dose and 3 incremental doses were recorded. After the last increment of 10 ?g?kg-1, the time for T1 to returned to 25% ,75% ,90% and TOF ratio(T4/T1) to 70% were recorded. The recovery index was also calculated.Results The demographic data were comparable between the 3 groups. The ED50 and ED95 were significantly lower in desflurane and isoflurane groups than those in control group(P 0.05 ) . The time for T1 to return to 25 % , 75 % and 90 % was significantly longer in desflurane and isoflurane group than that in the control group. The recovery from vecuronium-induced neuromuscular block was slower in desflurane group than that in isoflurane group( P

BACKGROUNDRas to MAPK pathway plays a critical role in the transmission of many growth and developmental signals. A new component of this pathway which is termed kinase suppressor of Ras (KSR) was found in 1995. KSR is as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase (MAPK) and its upstream regulators. It has been proven that KSR has many phosphorylation sites, and phosphorylation state changes response to signaling events. Site-directed mutagenesis can precisely change the base sequence and get mutant proteins. The aim of this study is to construct two mutant proteins of KSR by using site-directed mutagenesis, and to express and purify them, therefore to provide basement for studying the functional and biochemical mechanisms of KSR.

METHODSSite-directed mutagenesis of pCMV-Tag2b-KSR gene was performed by modified QuikChangtm site-directed mutagenesis kit method. Two pairs of mutagenic primers were synthesized in vitro and two mutations desired, the recombinant plasmids were verified by restriction enzyme analysis and DNA sequencing. Then positive clones were transfected into 293T cell line. The purified mutant proteins were analyzed by Western blot.

RESULTSTwo mutants were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant genes were completely concordant with experiment design, which could be used to be transfected into 293T cell line. The purified mutants were identified by Western blot.

CONCLUSIONSTwo mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.

AIM: To investigate the acute clinical manifestations of cosmetology-related ocular damage(COD).METHODS:Retrospective study. A total of 53 cases(89 eyes)with ocular damage caused by cosmetology from April 2016 to October 2021 were collected. The clinical features were analyzed, including age, gender, affected eye(s), clinical manifestations, injury cause, treatment procedures, and prognosis.RESULTS: All 53 patients were female, aged 22-45 years, with an average age of 28.4±6.7 years. Monocular injuries were observed in 17 patients, and binocular injuries in 36 patients. The same eye could exhibit two or more ocular damage simultaneously. The primary cosmetology procedures causing COD were eyeliner tattooing(38 eyes; 43%), eyelash extensions(18 eyes; 20%), removal of false eyelashes(11 eyes; 12%), mascara application(8 eyes; 9%), double eyelid surgery(6 eyes; 7%), and others(8 eyes; 9%). Major ocular damages included corneal damage(56 eyes; 63%), eyelid contact dermatitis(26 eyes; 29%), conjunctivitis(19 eyes; 21%), reactive eyelid edema(13 eyes; 15%), ocular surface foreign bodies(12 eyes; 14%), bacterial infection of the palpebral margin(10 eyes; 11%), and others(5 eyes; 6%). These 5 eyes included 1 eye(1%)with central retinal artery occlusion caused by periocular injection of hyaluronic acid. The majority of patients(74 eyes)recovered within 1-2 wk with appropriate treatment, while filamentosa keratitis appeared in 3 eyes and the eye with central retinal artery occlusion had poor prognosis.CONCLUSIONS: COD predominantly occurs in young and middle-aged females with cosmetology experience. The most common cosmetology procedure leading to COD is eyeliner tattooing, and corneal damage is the most significant type of COD. COD can be effectively prevented and treated, resulting in a generally favorable prognosis.

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Base Sequence ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUNDThe present experimental data have showed that the function of kinase suppressor of Ras (KSR) is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line.

METHODSN-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot.

RESULTSThe sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot.

CONCLUSIONSEukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.

BACKGROUNDPrevious researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.

METHODSSite-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.

RESULTSFive nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.

CONCLUSIONSFive nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.

BACKGROUNDThe results of our previous studies have proven that nm23-H1 gene can suppress metastasis of lung cancer, which may be associated with suppression of the Wnt signal pathway through up-regulating the activity of glycogen synthase kinase 3β (GSK-3β), the key kinase of the Wnt signal pathway. The aim of this study is to investigate the effect of point mutation of nm23-H1 gene on GSK-3β activity in cytoplasm and nucleus in human high-metastatic large cell lung cancer cell line L9981.

METHODSUsing immunoprecipitation and a radioactive isotope scintillation counter, the activity of GSK-3β was detected in cytoplasm and nucleus of human low-metastatic large cell lung cancer cell line NL9980, human high-metastatic large cell lung cancer cell line L9981, L9981-pEGFP (transfected with vector), L9981-nm23-H1-pEGFP (transfected with wild type nm23-H1), L9981-nm23-H1 S44A -pEGFP mutant (transfected with serine 44 to alanineon of nm23-H1 gene), L9981-nm23-H1 P96S -pEGFP mutant (transfected with proline 96 to serine of nm23-H1 gene), L9981-nm23-H1 H118F -pEGFP mutant (transfected with histidine 118 to phenylalanine of nm23-H1 gene) and L9981-nm23-H1 S120G -pEGFP mutant (transfected with serine 120 to glycine of nm23-H1 gene).

RESULTSThe GSK-3β activity in cytoplasm and nucleus was remarkably decreased in the transgene lung cancer cell lines transfected with mutant nm23-H1 cDNA (L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP). Significant differences of GSK-3β activity in cytoplasm and nucleus were observed (P < 0.05) when L9981-nm23-H1-pEGFP cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP lung cancer cell lines. There was a highly significant difference in GSK-3β activity in the cytoplasm between L9981-nm23-H1-pEGFP cell line and L9981-nm23-H1 P96S -pEGFP lung cancer cell line (P < 0.01 ). A highly significant difference in GSK-3β activity in the nucleus was observed (P < 0.01) when L9981-nm23-H1-pEGFP lung cancer cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP and L9981-nm23-H1 H118F -pEGFP lung cancer cell lines.

CONCLUSIONS(1) Point mutation of nm23-H1 gene can significantly influence the regulating effects on the GSK-3β activity in the cytoplasm and nucleus in the human high-metastatic large cell lung cancer cell line L9981. (2) The effects of nm23-H1 gene on metastatic phenotype may be related to the upregulation of GSK-3β activity in human high-metastatic large cell lung cancer cell line L9981.

BACKGROUNDScreening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study.

METHODSSubtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments.

RESULTSThe subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method.

CONCLUSIONSSSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.

BACKGROUNDTo clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell.

METHODShTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.

RESULTSElectrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line.

CONCLUSIONSThe hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

OBJECTIVETo observe the ultrastructural changes of sperm flagella in patients with severe idiopathic asthenospermia.

METHODSUsing the transmission electron microscope, we examined the ultrastructure of sperm flagella from 22 patients with severe idiopathic asthenospermia.

RESULTSUltrastructural anomalies were found in all the 22 patients, 6 with partial or complete absence of internal and external dynamic arms in dedicative of primary ciliary dyskinesia, 1 with hyperplasia, hypertrophy and disordered organization of the fibrous sheath usually referred to as dysplasia of the fibrous sheath, and the other 15 with non-specific flagellar anomalies.

CONCLUSIONExamination of the ultrastructure of sperm flagella in severe idiopathic asthenospermia patients can help to distinguish congenital from acquired flagellar structural anomalies and give valuable guidance in the treatment.