Objective To provide the experimental basis for the further research of the interacting proteins with Stathmin ,the Stathmin gene Pichia pastoris expression system was constructed ,the expressed Stathmin product was purified and identified .Meth‐ods Stathmin gene was amplified from tumor cell line of SKBR3 by PCR method and cloned into the yeast expression vector pPIC3 .5K .The recombinant vector pPIC3 .5K‐Stathmin was constructed and transformed into Pichia pastoris GS115 .The positive clones were screened by YPD medium containing Geneticin 600 μg/mL .Expression was induced with 0 .5% methanol and expres‐sion products were identified by SDS‐PAGE and Western Blotting .Results DNA sequencing result showed that the gene fragment was consistent with Stathmin gene sequence .pPIC3 .5K‐Stathmin was selected from YPD culture medium containing Geneticin ,and the positive clones were identified by PCR .SDS‐PAGE showed that a 37 × 103 protein band could be seen on the PAGE gel after Coomassie Blue staining ,which was further confirmed and identified as Stathmin protein by Western Blotting .Conclusion Stathmin yeast expression vector is successfully constructed and expressed in Pichia pastoris ,which laid the foundation for the study of inter‐acting proteins with Stathmin ,and for the preparation of the biological treatment drugs of Stahtmin target .

Objective To describe the immunological characteristics of refractory primary biliary cirrhosis compared with the typical patients for more than 1 year's administration of UDCA.Methods Sixty patients treated with UDCA for more than 1 year in our clinic were enrolled into this study.According to the response to UDCA by Paris criteria,patients were divided into refractory group (23 patients) and typical groups (37 patients).The recent peripheral lymphocyte subsets and cytokines of the two groups were tested and analyzed.One-way ANOVA and t test were used for statistical analysis.Results ① One-year treatment after diagnosis,there were no differences between the two groups in the distribution of peripheral lymphocytic subsets,meanwhile,the two groups had higher percentage of B cells,CD4+T cells,CD4+CD28+T cells and CD8+ CD28-T cells than healthy controls respectively.② The serum levels of IL-6 [(0.8±0.9) pg/ml vs (0.3±0.4) pg/ml] and HGF were higher in the refractory group than other groups.Conclusion During the plateau phase,refractory PBC patients have higher serum levels of IL-6 and HGF,which probably suggest that the refractory PBC patients may have severe immunologic disturbance in vivo.

Background Researches determined that the alteration of A69S locus of age-related maculopathy susceptibility 2 ( ARMS2 ) gene is closely associated with the pathogenesis and progression of age-related maculopathy ( AM D ).However,the location of ARMS2 protein in normal eye tissue is still in controversy,therefore,its function is below understanding up to now.Objective The goal of this laboratory work was to investigate the distribution,expression and location of ARMS2 protein in normal adult retina and choroid as well as in retinal pigment epithelial (RPE) cells and lay a basis for exploring further its function in the protein level.Methods Ten donor eyeballs of normal adult male with the age from 28-42 years were collected in eye bank of Qingdao Eye Hospital.The frozen sections of the retina and choroid were prepared for the detection and location of ARMS2 in 3 eyes by immunofluorescence under the confocal laser microscope.The retina was isolated for the primary culture of RPE cells using explant culture method.The cells were then identified by CK32 antibody by immunofluorescence.The distribution and expression of the ARMS2 protein in retina,ehoroid and RPE cells were determined by immunofluorescence technique.Results ARMS2 protein was strongly expressed in retinal vessel,RPE cell layer,Bruch membrane and choroidal vessel,but weak expression was in retinal ganglion cell layer,inner nuclear layer,outer plexiform layer,outer nuclear layer and inner plexiform layer in the normal eyes.The primarily cultured cells appeared the polygon shape with the abundant pigment in cytoplasm.The immunofluorescence of the cells showed the positive response for CK32,exhibiting the green fluorescence granules in the cytoplasm.The positive expression of ARMS2 protein also was seen in the cytoplasm of RPE cells,appearing the red fluorescence.Conclusions ARMS2 protein mainly distribute and locate retinal and choroidal vessels,RPE cells and Bruch membrane in normal eye.

Objective To explore the relationship between the Stathmin gene expression in breast cancer cells MDA-MB-231, MCF-7 and the biological behaviours such as cell growth,adhesion and invasion,and provide experimental basis of breast cancer metastasis for further study.Methods Used RT-PCR and Western Blot methods to detect the Stathmin gene expres-sion levels in MDA-MB-231 and MCF-7 cells,and in the mean while to test the MDA-MB-231 and MCF-7 cell growth,adhe-sion,invasion ability by CCK-8 cell proliferation experiments,cell adhesion experiments,cell invasion experiments,then, analyed the relationship of Stathmin gene expression and cell growth,adhesion,invasion ability.Results Over-expression levels of Stathmin gene were observed both in the MDA-MB-231 and MCF-7 cells (F=10.173,P<0.05),and furthermore, the expression levels of Stathmin gene in MDA-MB-231 cells was higher than in MCF-7 cells (t=4.562,P<0.05).While, the growth,adhesion and invasion ability of the MDA-MB-231 cells was higher than that of MCF-7 cells(P<0.05).Conclu-sion The higher level of Stathmin gene expression,the stronger breast cancer cells had ability of growth,invasion,and ad-hesive.The Stathmin gene expression levels was closely correlated with breast cancer cell invasive.

As the increasing population aging,the incidence of age-related macular degeneration is increasing,blinding rate also increasing,so it is very important for the early diagnosis and treatment of age-related macular degeneration.There are many methods to check the aging macular degeneration,such as fundus angiography,optical coherence tomography (OCT),visual field and multifocal electroretinogram (mfERG).In recent years,many emerging ophthalmic methods have emerged and are gradually applied to clinical diagnosis,including optic coherence tomography angiography (OCTA).The function of these methods has its unique advantages,but there are also limitations.This paper will review these existing methods.

The cyanuric chloride linkers have been used for cyclizing polypeptide, but not used for α-conotoxin, the peptides with rich disulfide bonds and more amino acid residues. In this study, cyclic peptides c[A10L]PnIA-1-4 were synthesized efficiently by lysine assisted cyanuric chloride linkers with 28.92%-52.00% yields. The activity evaluation showed that the IC₅₀ values of c[A10L]PnIA-1 against α7 and α3β2 nAChR subtypes were 5 and 7 times higher than [A10L]PnIA respectively, and the subtype selectivity was maintained. The results of circular dichroism show that this cyclization method had no significant effect on its secondary structure. Compared with the commonly used head-to-tail cyclization in conotoxin cyclization, this method has the advantages of rapid reaction and high yield, which is expected to be further applied to the cyclization study of various α-conotoxins.

Adolescent ; Adult ; Anemia, Aplastic ; etiology ; therapy ; Benzene ; poisoning ; Bone Marrow Cells ; drug effects ; pathology ; Female ; Humans ; Male ; Occupational Diseases ; etiology ; therapy ; Occupational Exposure ; adverse effects

Background:The platelet to lymphocyte ratio (PLR) has recently emerged as a potential inflammatory biomarker and has been shown to be significantly associated with atherosclerotic coronary artery disease (CAD).Therefore,we aimed to explore the association of PLR with in-hospital major adverse cardiovascular events (MACEs) and the severity of CAD assessed by the Gensini score (GS) in patients with acute myocardial infarction (AMI) undergoing coronary angiography.Methods:A total of 502 patients with AMI consecutively treated at the Affiliated Hospital of Qingdao University (Qingdao,China) and underwent coronary angiography from August 2017 to December 2018 were recruited in this study.The demographic,clinical,angiographic characteristics,and laboratory parameters were collected.According to the presence of in-hospital MACEs,the included patients were divided into the MACE group (n =81) and the non-MACE group (n =421).Further,according to tertiles of the GS,the patients were classified into three groups:the low GS group (GS ≤ 32 points,n =173),medium GS group (32 points ＜ GS ≤ 60 points,n =169),and high GS group (60 points ＜ GS ≤ 180 points,n =160).The main statistical methods included Chisquared test,non-parametric Mann-Whitney U test,Kruskal-Wallis H test,logistic regression,and receiver operating characteristic curves.Results:The PLR in the MACE group was significantly higher than that in the non-MACE group (179.43 [132.84,239.74] vs.116.11 [87.98,145.45],Z =-8.109,P ＜ 0.001).Further,there were significant differences in PLR among the tertiles of GS (110.05 [84.57,139.06] vs.119.78 [98.44,157.98] vs.140.00 [102.27,191.83],H=19.524,P ＜ 0.001).PLR was demonstrated to be an independent risk factor of in-hospital MACEs (odds ratio [OR]:1.012,95 ％ confidential interval [CI]:1.006-1.018,P ＜ 0.001) and severe CAD assessed by the GS (OR:1.004,95％ CI:1.002-1.009,P =0.042).The cutoff value of PLR for predicting the development of in-hospital MACEs was 151.28 with a sensitivity of 66.7％ and a specificity of 78.1％ (area under the curve [AUC]:0.786,95％ CI:0.730-0.842,P ＜ 0.001),and a PLR of 139.31 was also identified to be an effective cutoff point for detecting a high GS (＞60 points) with a sensitivity of 49.4％ and a specificity of 69.6％ (AUC:0.611,95％ CI:0.556-0.666,P ＜ 0.001).Conclusions:PLR as a novel inflammatory marker is significantly and independently associated with the occurrence of in-hospital MACEs and the severity of CAD assessed by the GS in patients with AMI.As an easily available and inexpensive inflammatory indicator,PLR could be widely used as an efficient inflammatory biomarker for identifying high-risk patients and for individualizing targeted therapy to improve the prognosis of AMI.

AIMTo investigate the effects of L-arginine and nitric oxide synthase (NOS) inhibitor Aminoguanidine (AG) on endotoxin induced lung injury in rats.

METHODSForty eight healthy male SD rats weighing (300 +/- 20) g were used. The animals were anesthetized with 20% urethane 1 g x kg(-1). Common carotid artery (CAA) and common carotid vein (CAV) were exposed through a median incision in the neck. Mean arterial pressure (MAP) was measured through a pressure transducer connected with intubation of CAA. The animals were randomly divided into six groups: group 1: control: group 2: LPS (5 mg x kg(-1) intravenous injection, i.v.); group 3: AG (50 mg x kg(-1) intraperitoneal injection, IP); group 4: high dose L-arginine (500 mg x kg(-1), IP); group 5: low dose L-arginine (250 mg x kg(-1) IP). Group 6: L-arginine + AG (250 mg x kg(-1), 50 mg x kg(-1), IP). Group 1: The animals were killed 6 h after 0.9% saline solution was given. Group 2: 0.9% saline solution was given 3 h after LPS i.v. and the animals were killed 3 h after medication. Group 3, 4, 5 and 6: AG, L-arginine and L-arginine+ AG were given 3 h after LPS i.v. respectively and the animals were killed 3 h after medication respectively. The pulmonary was removed immediately. The pulmonary coefficient and water content in pulmonary tissue were calculated (%). The NO content in plasma, MDA content and NOS, SOD activity in the pulmonary tissue were measured.

RESULTSL-arginine, AG and L-arginine + AG significantly decreased pulmonary coefficient and water content in pulmonary tissue and ameliorated endotoxin induced lung injury. AG and L-arginine + AG significantly decreased NO content in plasma, decreased MDA content and inhibited NOS activity and enhanced SOD activity in the pulmonary tissue.

CONCLUSIONIt may be concluded that L-arginine, AG and L-arginine + AG have beneficial effects on lung injury induced by LPS.

Animals ; Arginine ; administration & dosage ; therapeutic use ; Endotoxins ; adverse effects ; Guanidines ; administration & dosage ; therapeutic use ; Lung Injury ; chemically induced ; drug therapy ; Male ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

OBJECTIVESTo investigate the role of glycyrrhizin on TGFbeta1 stimulated signaling transduction in rat hepatic stellate cells (HSCs).

METHODSThe mice HSCs were isolated and cultured with or without glycyrrhizin (1 micromol/L-1000 micromol/L) in vitro after TGFbeta1 stimulation. The mRNA level of Smad2, 3, 7 were measured with RT-PCR; protein expression level of Smad2, 3, 7 and collagen I, III were analyzed with Western blot.

RESULTSTGFbeta1 increased the mRNA level and protein expression of Smad2, 3, 7 in HSC; it also increased protein expression of collagen I and III. 1 micromol/L-1000 micromol/L glycyrrhizin decreased the mRNA level and protein expression of Smad2, 3, 7; it also inhibited protein expression of collagen I and III gradually.

CONCLUSIONInterventing the TGFbeta signaling pathway and decreasing the synthesis of collagen, might be involved in the anti-fibrosis mechanism of glycyrrhizin.

Animals ; Glycyrrhizic Acid ; pharmacology ; Hepatocytes ; metabolism ; Male ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; pharmacology