BACKGROUND: Small conductance,Ca2+-activated potassium(SK)channel,presents in various cell types and plays a crucial role in action potential profile.However,coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene,so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2.After being identified,they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors.The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation,and their activation was tested.The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411,546 and 729 bp,respectively.Three sub-clones of pGBKT7-SK2 were successfully constructed.Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements.The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated.The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.

BACKGROUND: Chronic obstructive pulmonary disease(COPD) is characterized of chronic inflammation in airway, pulmonary parenchyma and pulmonary vessels. The mechanism of the increment and activity changes of these inflammatory cells is unclear at present.OBJECTIVE: To study the apoptotic character of peripheral blood polymorphonuclear neutrophils(PMNs) and its relationship with COPD to provide a reference for early intervention and function surveillance for COPD patients.DESIGN: An observatory comparative study based on COPD patients and healthy population as controls.SETTING: Department of pulmonary medicine in a military medical university of Chinese PLA affiliated hospital.PARTICIPANTS: Totally 98 COPD patients were admitted by the Department of Pulmonary Medicine, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between February 2003 and December 2003 due to COPD acute attack. Eighteen patients including 12 males and 6 females aged between 48 and 70 years old [mean of(56 ± 7) years old]were randomly selected into COPD group according to random number table.Totally 14 healthy adults including 10 males and 4 females aged between 50 and 70 years old [mean of (59 ± 8) years old] who were individuals came for physical check up in our hospital were selected in control group.METHODS: PMNs were separated from peripheral blood by density gradient centrifugation. The dynamic changes of PMNs apoptosis in peripheral blood was observed by flow cytometer and TUNEL method.MAIN OUTCOME MEASURES: Comparison of PMNs apoptotic rate in peripheral blood among groupsRESULTS: As indicated by flow cytometric analysis, PMNs apoptotic rate at early apoptotic phase in COPD patients at paracmasis was(8.5 ± 1.3)%,which was significantly lower than(12.5 ± 1.8)% of normal control group( t=6.25, P ＜ 0. 01); PMNs apoptotic rate was(5.1 ±0.6)% at acute aggravation stage, which was significantly lower than that of paracmasis group ( t =5.66, P ＜001). As indicated by TUNEL analysis, PMNs apoptotic rate at paracmasis was(12.42 ±2.7)% , which was significantly lower than (21.5±4.8)% of normal control group(t=5.76, P ＜ 0.01); PMNs apoptotic rate was(4. 9 ±0.4)% at acute aggravation stage, which was significantly lower than that of paracmasis group( t = 6. 12, P ＜ 0. 01 ) . PMNs changes at the late phase of apoptosis/necrosis had a contrary tendency, i. e.,PMNs rate at late apoptotic phase/necrosis was(2. 8 ± 0.5)% in COPD patients at paracmasis, which was significantly higher than(1. 3 ±0.4)% of normal control group ( t= 6. 37, P ＜ 0. 01 ); PMNs rate was (3.7 ± 0. 3) % at acute aggravation stage, which was significantly higher than that of paracmasis group(t=5.81, P ＜0.01).CONCLUSION: PMNs abnormal apoptosis might be an important reason that induces PMNs aggregation in airway and lung tissues in COPD. This process might have an important significance in the generation and development of chronic airway inflammation, which provides an etiologic basis for the primary rehabilitative intervention of COPD.

Objective To study the expression of Gq protein in bronchopulmonary tissues of rats of chronic obstructive pulmonary disease (COPD) model and the roles of inflammation in airway of COPD rats. Methods A total of 44 Wistar rats were divided randomly into 4 groups: nonsmoking control group, 1 month smoking group, 2 month smoking group, and intervention group (dexamethasone + smoking for 2 months). The pathological changes of lung tissues and the total and differential count of white blood cells in bronchoalveolar lavage fluid (BALF) in all groups were observed. The expression of Gq in bronchopulmonary tissues was detected by immunohistochemical method and Western blot analysis. Results More significant increases in total number of leukocytes and neutrophils in BALF were found in the model group than those in the control group. Gq was mainly expressed in airway epithelial cells, inflammatory cells, and pulmonary capillary endothelia. Weak expression of Gq was found in bronchopulmonary tissues in the control group, but overexpression of Gq in the model group. The ratios of Gq positive cells in bronchopulmonary tissues were significantly correlated with the number of white blood cells and neutrophils in BALF. The levels of expression of Gq were significantly higher in the model groups and intervention group than those in the control group. More significant decrease in Gq expression was found in the intervention group as compared with that in 3 month smoking group. Conclusion Gq may be involved in the process of airway inflammation in COPD. Inhibition of the Gq overexpression might be a new approach to the treatment of COPD. Corticosteroid might have some effects on the inhibition of the airway inflammation.

AIM:To study whether cordycepin (Cordy) plays a role in myocardial protection by regulating the expression of microRNA-455 ( miR-455) and reducing apoptosis induced by endoplasmic reticulum stress in the ischemia -reperfusion (IR) rats.METHODS: SD rats (250 ~300 g) were randomly divided into 3 groups: control group, the chests of the rats were only opened;IR group, the rats were given myocardial ischemia for 30 min, and then reperfusion for 120 min;IR+Cordy group:before IR, the rats were given Cordy (10 mg/kg) through femoral vein injection once a day for one week, and the last injection was given 30 min before ischemia.Automated biochemical analysis was used to detect rat serum activity of LDH and CK-MB.The myocardial endothelial cell ( EC) apoptotic rate was measured by TUNEL , and the ultrastructural changes of the myocardial EC were observed under transmission electron microscope .RT-qPCR was used to detect the expression of miR-455 and the mRNA levels of glucose-regulated protein 78 ( Grp78) and caspase-12 in the myo-cardium.RESULTS:Compared with control group, EC apoptosis in IR group was significantly increased (P<0.05). Compared with IR group , EC apoptosis in IR +Cordy group decreased significantly ( P<0.05 ) .Compared with control group, swelling of mitochondria , irregular membrane , loose wrinkles with vacuoles , disappeared matrix granules , irregular nuclear membrane , chromatin condensation , disappeared nucleoli and even small apoptosis body in the EC were found in IR group.Compared with IR group , the symptoms in IR +Cordy group were greatly improved .Compared with control group, the mRNA expression of Grp78 and caspase-12 as well as the miR-455 level in IR group was increased (P<0.05). Compared with IR group , the mRNA expression of Grp78 and caspase-12, as well as the miR-455 level in IR+Cordy group was decreased (P<0.05).CONCLUSION:Cordycepin attenuates myocardial EC apoptosis , down-regulates the expres-sion of miR-455, and inhibits endoplasmic reticulum stress in the myocardium .

Objective To analyze endoscopic ultrasonography (EUS) image of esophageal granular cell tumor (GCT) by computer image analysis software,and to explore its diagnostic and differential diagnostic value.Methods Eight esophageal GCT and eight leiomyomas confirmed by pathological and immunohistochemical examination were collected,mean gray value and gray value standard deviation of EUS image of the leisons were analyzed by computer image analysis software.The comparison of two groups was analyzed by t test.Results The endoscopic and ultrasound images of esophageal GCT and leiomyomas were similar.The mean gray value of EUS image of esophageal GCT and esophageal leiomyomas was 67.51 and 51.76,the difference was statistically significant (t=2.225,P=0.043).However,there was no significant difference in the standard deviation of gray value (13.54 vs 13.12,t=0.225,P=0.825).Conclusion The accuracy of GCT diagnosed by EUS could be increased by comparing the gray values of EUS images of esophageal GCT and leiomyomas.

Objective To investigate the protective effect and the possible mechanism of intravenous infusion with perfluorocarbon pretreatment and posttreatment on acute lung injury in rats on LPS-induced acute lung injury in rats.Methods A total of 24 Wistar rats were randomly divided into 4 groups:control group(NS group),LPS group,Pre group and Post group.Normal saline was given to NS group as a control.Rats were treated with LPS by intratracheal instillation in LPS group,rats received PFC through femoral vein.prior to LPS instillation in Pre group and rats received PFC through femoral vein after LPS instillation in Post group.NS group were sacrificed at 6 h after being injected with NS,LPS group,Pre group and Post group were sacrificed at 6h after being given LPS.Pathological changes of king,PaO2,lung wet to dry weight ratio (W/D),expression of myeloperoxidase (MPO) and intercellular adhesion molecule-1 (ICAM-1) of lung were assessed.Results Intravenous.infusion with perfluorocarbons increased PaO2,decreased W/D,MPO and ICAM-1 significantly (P＜0.05),and this effect is more remarkable in Pre group.Conclusion Intravenous infusion of PFC significantly protects lung from acute lung injury,especially by pretreatment forms,probably by down-regulate the expression of ICAM-1.

Objective: The study was designed to evaluate the changes and significance of circulating CD4~+CD25~+ and CD8~+CD28~- regulatory T cells (Tregs) in patients with multiple myeloma (MM).Methods:CD4~+CD25~+ and CD8~+CD28~-Tregs in peripheral blood of 38 patients with MM and of 20 healthy doners were measured by flow cytometry.Serum albumin and β_2-MG in patients with MM were measured using bromocresol green method,transmission turbidimetry respectively.Results:Compared to those of the controls,the proportions of CD4~+CD25~(+/high),CD4~+CD25~(high) CD127~(low) and CD8~+CD28~-Treg cells in newly diagnosed MM patients were elevated.Furthermore,the proportions of CD4~+CD25~(high) and CD4~+CD25~(high)CD127~(low) Tregs in each clinical stage were elevated when compared to those of the controls.The number of the Tregs were increasing with clinical stages and were significantly higher in stage Ⅲ MM than in stageⅠ MM;In stageⅡand Ⅲ MM,there were also elevated proportions of CD8~+CD28~- Tregs,increasing with clinical stages.However,there were no differences when compared between stage Ⅰ MM and the controls;Both the proportions of CD4~+CD25~(+/high) and CD4~+CD25~(high)CD127~(low) Tregs in active MM were not different from stable MM,although all of them were higher than those of controls.The proportion of CD8~+CD28~- Tregs was higher in active MM than in stable MM and controls,but there were no differences when compared between active and stable MM.The proportions of both CD4~+CD25~(high) Tregs and CD4~+CD25~(high)CD127~(low)Tregs had negative correlation with the levels of serum albumin.Conclusion:MM patients have elevated levels of circulating CD4~+CD25~+ and CD8~+CD28~-Tregs,which may be an important mechanism of MM immune evasion,and may be associated with clinical stages,disease progression and prognosis of MM to some extent.

Objective To investigate the diagnostic value of endoscopic ultrasonography (EUS) for duodenal cysts.Methods Clinical manifestations, results of conventional gastroscopy and EUS, as well as follow-up, of patients with duodenal cysts were retrospectively analyzed.Results A total of 105 patients with duodenal cysts presented with no lesion-related symptoms, but displayed characteristic imaging changes under EUS, which could reveal the origin, size and nature of duodenal cysts, and differentiate it from other submucosal lesions.A maximum follow-up of 5 years showed no changes in duodenal cysts.Conclusion Duodenal cyst is a benign disease, and EUS plays a very important role in diagnosis of the disease.

Adult ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Non-alcoholic Fatty Liver Disease ; pathology ; Obesity ; pathology ; Young Adult

A total of 105 patients with first-episode schizophrenia (male =51,female =54) and 99 normal controls (male =51,female =48) were included into this retrospective case-control study.Childhood trauma questionnaire-28 item short form (CTQ-SF) was used to assess the experience of childhood abuse.The result of binary logistic regression showed that emotional abuse (β =0.630,P ＜ 0.05) and emotional neglect(β =0.270,P ＜ 0.05) were included into the final model of predicting schizophrenia.It indicates that patients with first-episode schizophrenia experienced more early life stress than controls.Particularly emotional abuse and emotional neglect may play important roles in the onset of schizophrenia.