OBJECTIVETo construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line.

METHODSAn expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recombined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 microg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits.

RESULTSImmune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day.

CONCLUSIONHuman insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

Animals ; Cell Line ; Dose-Response Relationship, Drug ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; genetics ; Insulin ; biosynthesis ; genetics ; Mice ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Proinsulin ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

OBJECTIVETo evaluate the emotional and cognitive status in patients with obstructive sleep apnea syndrome (OSAS), using neuropsychological tests and evoked-related potential (P3).

METHODSSixteen patients diagnosed of OSAS were tested by Hamilton rating scale for anxiety (HRSA) and Hamilton rating scale for depression (HRSD). Other three groups, OSAS patient group (n = 21), snoring group (n = 21), and control group (n = 21), were administered polysomnography (PSG), auditory evoked event-related potential (P3), and clinic memory test. The results were analyzed using general linear model (GLM) analysis and Post Hoc test.

RESULTSTwelve OSAS patients' scores of HRSA and HRSD were beyond the normal range, 26.42 +/- 4.48 and 22.08 +/- 3.97 respectively. The auditory P3 latency in OSAS group was 363.1 +/- 22.9 ms (Fz), 368.57 +/- 28.03 ms (Cz), in snoring group 336.57 +/- 31.08 ms (Fz), 339.81 +/- 31.76 ms (Cz), in control group 340.8 +/- 28.7 ms (Fz), 338.29 +/- 29.21 ms (Cz). There were significant differences between OSAS group and snoring group, as well as control group (P < 0.05). No significant difference was seen between snoring group and control group. No significant difference was noted in P3 amplitude among three groups. Memory quotient (MQ) reduced in snoring group compared with control group.

CONCLUSIONSEmotional disturbances are common clinical features in OSAS patients. Abnormal auditory P3 latency indicates the cognitive dysfunction in OSAS patients. Nocturnal hypoxaemia may play an important role on it. Snorers should be monitored because of the tendency to develop cognitive impairment.

Adult ; Age Distribution ; Cognition Disorders ; physiopathology ; Depression ; physiopathology ; Evoked Potentials, Auditory ; Female ; Humans ; Male ; Middle Aged ; Neuropsychological Tests ; Polysomnography ; methods ; Sleep Apnea, Obstructive ; physiopathology ; psychology ; Snoring ; physiopathology

OBJECTIVETo confirmed reliability and feasibility of intranasal nerve growth factor (NGF) bypassing the blood-brain barrier and its potential neuroprotective effects on acute cerebral ischemia.

METHODS(1) To assay NGF concentrations in different brain regions after middle cerebral artery occlusion (MCAO). Rats were randomly divided into intranasal (i.n.) NGF, intravenous (i.v.) NGF, and untreated group (n = 4). The concentrations of NGF of different brain regions in the three groups after MCAO were measured by ELISA. (2) To observe neuroprotective action of NGF on focal cerebral ischemic damage. Rats were randomly assigned to 4 groups: i.n. vehicle, i.n. NGF, i.v. vehicle, i.v. NGF (n = 8). Treatment was initiated 30 minutes after onset of MCAO and given again 24 hours later. Three neurologic behavioral tests were performed 24 and 48 hours following onset of MCAO. Corrected infarct volumes were determined 48 hours after onset of MCAO.

RESULTSThe olfactory bulb in i.n. NGF group obtained the highest concentration (3252 pg/g) of NGF among all regions, followed by the hippocampus. The NGF concentrations in the olfactory bulb and hippocampus in i.n. NGF group were markedly higher than that in i.v. NGF and control groups. The infarct volume in i.n. NGF group was markedly reduced by 38.8% compared with i.n. vehicle group. I.n. NGF group vestibulum function markedly improved compared with i.n. vehicle group at 24 and 48 hours after onset of MCAO (P24h = 0.02 and P48h = 0.04, respectively).

CONCLUSIONIntranasal NGF could pass through the blood-brain barrier, reach the central nervous system, reduce infarct volume, and improve neurologic function in rats following MCAO. Intranasal delivery of NGF may be a promising treatment for stroke.

Administration, Intranasal ; Animals ; Behavior, Animal ; drug effects ; Blood-Brain Barrier ; Brain ; metabolism ; pathology ; Hippocampus ; metabolism ; Infarction, Middle Cerebral Artery ; metabolism ; pathology ; Injections, Intravenous ; Male ; Nerve Growth Factor ; metabolism ; pharmacology ; Neuroprotective Agents ; pharmacology ; Olfactory Bulb ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study endothelial damage by observing changes of circulating endothelial cells (CECs) in blood, coagulation and fibrinolysis index in patients with acute respiratory distress syndrome.

METHODSCECs were separated by isopycnic centrifugation method in 14 patients with acute lung injury (ALI), 7 patients with acute respiratory distress syndrome (ARDS), 10 intensive care unit (ICU) controls, and 15 healthy controls. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FG), fibrin degradation products (FDP), and D-dimer were examined simultaneously. Acute physiology and chronic health evaluation (APACHE) II and lung injury score (LIS) were recorded to evaluate severity of illness and lung injury.

RESULTS(1) The number of CECs in ALI (10.4 +/- 2.3) and ARDS groups (16.1 +/- 2.7) was higher than that in the healthy (1.9 +/- 0.5) (P < 0.01). In both ALI and ARDS, the number of CECs correlated with APACHE II (r = 0.55, P < 0.05 and r = 0.62, P < 0.05, respectively) and LIS (r = 0.60, P < 0.05 and r = 0.53, P < 0.05, respectively). CEC number was negatively correlated with PaO2 in ALI and ARDS (r = -0.49, P < 0.05 and r = -0.64, P < 0.05, respectively). (2) The level of FDP and D-dimer were higher in ALI and ARDS patients than that in ICU and healthy control groups (P < 0.05). The level of FG in ARDS group was significantly higher than in the ICU and healthy control groups (P < 0.05). But in ALI group, the level of FG was significantly higher than only healthy control group (P < 0.05).

CONCLUSIONSEndothelial cell damage occurs in ARDS patients, which may play a major role in the pathophysiology of ARDS. Changes of endothelial cell activation and damage markers, such as CECs, plasma coagulation and fibrinolysis index, to some extent reflect severity of illness and lung injury in ARDS.

APACHE ; Adult ; Aged ; Blood Coagulation ; Cell Count ; Endothelial Cells ; pathology ; Female ; Fibrin Fibrinogen Degradation Products ; metabolism ; Fibrinogen ; metabolism ; Humans ; Male ; Middle Aged ; Partial Thromboplastin Time ; Prothrombin Time ; Respiratory Distress Syndrome, Adult ; blood ; pathology

OBJECTIVETo observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.

METHODSSalt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.

RESULTSAfter 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7 +/- 12.1 mmHg vs. 2.8 +/- 5.2 mmHg, P < 0.01; 12.2% +/- 12.0% vs. 2.5% +/- 4.4%, P < 0.001, respectively). There was a similar trend for diastolic blood pressure (8.4 +/- 6.4 mmHg vs. 3.7 +/- 6.4 mmHg, P = 0.052; 13.2% +/- 10.6% vs. 6.8% +/- 10.1%, P = 0.053, respectively).

CONCLUSIONSSalt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.

Adolescent ; Aging ; physiology ; Blood Pressure ; drug effects ; Blood Volume ; Female ; Furosemide ; pharmacology ; Humans ; Infusions, Intravenous ; Male ; Sodium Chloride ; administration & dosage ; pharmacology ; Systole

OBJECTIVETo investigate the effect of peroxisome proliferator-activated receptor-alpha (PPAR alpha) and PPAR gamma activators on tumor necrosis factor-alpha (TNFalpha) expression in neonatal rat cardiac myocytes.

METHODSPrimary cultures of cardiac myocytes from 1- to 3-day-old Wistar rats were prepared, and myocytes were exposed to lipopolysaccharide (LPS) and varying concentrations of PPAR alpha or PPAR gamma activator (fenofibrate or pioglitazone). RT-PCR and ELISA were used to measure TNFalpha, PPAR alpha, and PPAR gamma expression in cultured cardiac myocytes. Transient transfection of TNFalpha promoter with or without nuclear factor-kappaB (NF-kappaB) binding site to cardiac myocytes was performed.

RESULTSPretreatment of cardiac myocytes with fenofibrate or pioglitazone inhibited LPS-induced TNFalpha mRNA and protein expression in a dose-dependent manner. However, no significant changes were observed on PPAR alpha or PPAR gamma mRNA expression when cardiac myocytes were pretreated with fenofibrate or pioglitazone. Proportional suppression of TNFalpha promoter activity was observed when myocytes was transiently transfected with whole length of TNFalpha promoter (-721/+17) after being stimulated with LPS and fenofibrate or pioglitazone, whereas no change of promoter activity was observed with transfection of TNFalpha reporter construct in deletion of NF-kappaB binding site (-182/+17).

CONCLUSIONSPPAR alpha and PPAR gamma activators may inhibit cardiac TNFalpha expression but not accompanied by change of PPAR alpha or PPAR gamma mRNA expression. Therefore PPAR alpha and PPAR gamma activators appear to play a role in anti-inflammation. The mechanism may partly be involved in suppression of the NF-kappaB pathway.

Animals ; Animals, Newborn ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fenofibrate ; pharmacology ; Lipopolysaccharides ; pharmacology ; Myocytes, Cardiac ; metabolism ; NF-kappa B ; metabolism ; PPAR alpha ; biosynthesis ; genetics ; PPAR gamma ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Thiazolidinediones ; pharmacology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

Animals ; Apoptosis ; drug effects ; Male ; Malondialdehyde ; blood ; Myocardial Ischemia ; blood ; pathology ; Myocardial Reperfusion Injury ; blood ; pathology ; Myocytes, Cardiac ; pathology ; Protective Agents ; pharmacology ; Rabbits ; Random Allocation ; Superoxide Dismutase ; blood ; Trimetazidine ; pharmacology

OBJECTIVETo explore the effects of mifepristone on the growth of human gastric cancer cell line MKN-45 and its possible mechanisms.

METHODSIn situ hybridization was used to detect the expression of progesterone receptor (PR) mRNA in MKN-45 cells. Proliferation, cell cycle distribution, and the expression of Bcl-xL and vascular endothelial growth factor (VEGF) of MKN-45 cells incubated with various concentrations of mifepristone (1, 5, 10, and 20 micromol/L) were analyzed using MTT reduction assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoabsorbent assay (ELISA), respectively. After transplantation of MKN-45 cells underneath the skin of athymic mice, mifepristone was administrated with the dose of 50 mg/(kg x d) for 6 weeks to evaluate the tumor growth. Apoptosis and the expression of proliferating cell nuclear antigen (PCNA) in xenografted tumors were detected using transmission electron microscopy and immunohistochemical staining, respectively.

RESULTSPR mRNA was highly expressed in cultured MKN-45 cell. Mifepristone dose-dependently inhibited the proliferation of MKN-45 cells, and the inhibitory rate was dramatically increased from 7.21% to 47.23%. The inhibitory effect was accompanied by a dose-dependent increase in the percentage of cells in G0/G1 phase, and with a concurrent decrease in the proportion of S- and G2M-phase cells and the proliferative index from 57.65% to 24.54%. Meanwhile, mifepristone down-regulated the expression of Bcl-xL and VEGF in a dose-dependent manner. In vivo, mifepristone effectively inhibited the growth of xenografted tumors in nude mice (55.14% for inhibitory rate), induced apoptosis, and down-regulated PCNA expression in gastric cancer.

CONCLUSIONMifepristone exerts significant growth inhibitory effects on PR-positive human MKN-45 gastric cancer cells via multiple mechanisms, and may be a beneficial agent against the tumor.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mifepristone ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; biosynthesis ; genetics ; Stomach Neoplasms ; metabolism ; pathology

Adult ; Convergence, Ocular ; physiology ; Eyeglasses ; Female ; Humans ; Male ; Myopia ; physiopathology ; Vision, Binocular ; physiology

Adult ; Echocardiography, Three-Dimensional ; methods ; Heart Ventricles ; diagnostic imaging ; Humans ; Stroke Volume ; physiology ; Ventricular Function ; Ventricular Function, Left ; Ventricular Function, Right