As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

Malignant melanoma still remains to be a serious health threat. Overexpression of focal adhesion kinase (FAK) in melanoma has suggested that FAK could be a promising target for therapeutic intervention. To further investigate the function of FAK in melanoma, FAK expression was down-regulated by stable transfection of plasmid harboring FAK small interfering RNA (siRNA) into melanoma cell line. Two stable cell lines, F10-siFAK and F10-control, have been constructed and screened. Compared with the F10-control, both the mRNA and protein levels of FAK decreased significantly, and the cell cycle of F10-siFAK was arrested at G1 phase. Furthermore, the tumor growth rate of F10-siFAK cells was notably slower than that of F10-control in in vivo tumor models. These results show that FAK is an important regulatory gene in melanoma. The stable FAK-knockdown melanoma cell line is an useful tool for further investigation of FAK's function in the progression of melanoma, and also an effective means of drug screening for anti-melanoma therapeutics.
Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; G1 Phase ; Gene Knockdown Techniques ; Melanoma, Experimental ; enzymology ; pathology ; Mice ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Plasmids ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

Objective The purpose of this paper is to screen inborn errors of meta bolism (IEM) by analyzing urinary components, so as to provide laboratory guide for their diagnosis and therapy.Methods Urine samples of patients suspected to have IEM were collec- ted.Urea was de compo sed with urease and n-heptadecanoic acid was added as internal standard.Protein was denatured with ethanol and precipitate was removed by centrifugation,dried b y evaporation, the residue was trimethylsilylly derivatized with BSTFA/TMCS,and then analyzed with GC-MS for quantification of organic acids, amino acids,suga rs, polyols, purines and pyrimidines, simultaneously. This procedure is denom inated as urease pretreatment-gas chromatography-mass spectrometry (UP-GC-MS) internationally.Results Urinary samples of 327 patients from 6 provinces, cities and autonomous regions were analyzed,and 16 kinds of 27 cases of IEM were screened out with a positiv e rate of 8.26%,among which there were 3 cases of hyperphenylalaninemia,3 cases of glyceroluria,3 cases of Leigh syndrome, 2 cases of propionic acidemia, 2 case s of methylmalonic aciduria, 2 cases of von Gierke′s disease, 2 cases of fructo se-1,6-diphosphatase deficiency, 2 cases of fructosuria, 1 cases of multiple car boxylase deficiency, 1 cases of glutaric acidemia typeⅠ, 1 cases of maple sy rup urine disease, 1 cases of hyperglycinemia, 1 cases of 3-aminoisobutyric acid uria,1 cases of adult-onset typeⅡcitrullinemia,1 cases of galactosemia and 1 ca ses of Fanconi′s syndrome.Several IEM patients above had died,but satisfactory therapeutic effects had been achieved in some diseases,in cluding multiple carboxylase deficiency,methylmalonic aciduria and galactosemia. Other patients′ condition remained to be followed up.Conclusion Analysis of urinary components by UP-GC-MS provides a valuable tool for screenin g of IEM and the results will help to provide effective diagnostic and therapeut ic guide for the patients. J Appl Clin Pediatr,2005,20(2):142-144

Objective To study the effect of exogenous prostaglandin E 1 (PGE 1) on the superoxide dismutase(SOD) and nitric oxide(NO) levels in brain tissue of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Sixty 7-day old newborn Wistar rats to establish HIBD models,intraperitoneally and subcutaneous injected PGE 1 and TMP,then the rats were killed after hypo- xia and ischemia for 48 hours.Take cerebral cortex of arteria carotis ligation side and made them into homogenate to detect SOD and NO levels in brain tissue.Results SOD level in HIBD group was lower,and NO level was higher than those of normal group(P

BACKGROUNDIn tumors the process of apoptosis occurs over an interval of time after chemotherapy. It is important to determine the best time for detecting apoptosis by in vivo imaging. In this study, we evaluated the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using a (99)Tc(m)-labeled Annexin V recombinant with ten consecutive histidines (His10-Annexin V) in a mouse model.

METHODS(99)Tc(m)-His10-Annexin V was prepared by one step direct labeling; radio-chemical purity (RCP) and radio-stability was tested. The binding of (99)Tc(m)-His10-Annexin V to apoptotic cells was validated in vitro using camptothecin-induced Jurkat cells. In vivo bio-distribution was determined in mice by dissection. The human H460 NSCLC tumor cell line (H460) tumor-bearing mice were treated with intravenous paclitaxel 24, 48 and 72 hours later. (99)Tc(m)-His10-Annexin V was injected intravenously, and planar images were acquired at 2, 4 and 6 hours post-injection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and they reflected specific binding of (99)Tc(m)-His10-Annexin V. Mice were sacrificed after imaging. Caspase-3, as the apoptosis detector, was determined by flow cytometry, and DNA fragmentation was analyzed by the terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Nonspecific accumulation of protein was estimated using bovine serum albumin (BSA). The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity.

RESULTS(99)Tc(m)-His10-Annexin V had a RCP > 98% and high stability 2 hours after radio-labeling, and it could bind to apoptotic cells with high affinity. Bio-distribution of (99)Tc(m)-His10-Annexin V showed predominant uptake in kidney, relatively low uptake in myocardium, liver and gastrointestinal tract, and rapid clearance from blood and kidney was observed. The T/NT was significantly increased after paclitaxel treatment, whereas it was low in untreated tumors (T/NT = 1.43 ± 0.18). The %ID/g activity in Group 2 (24 hours), Group 3 (48 hours) and Group 4 (72 hours) after treatment was 2.55 ± 0.73, 3.35 ± 1.10, and 3.4 ± 0.96, respectively. Whereas in the non-treated group, Group 1, %ID/g was 1.10 ± 0.18. The radiotracer uptake was positively correlated to the apoptotic index (r = 0.852, P < 0.01), as well as caspase-3 activity (r = 0.816, P < 0.01).

CONCLUSIONThis study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using (99)Tc(m)- His10-Annexin V.

Animals ; Annexin A5 ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Histidine ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Organotechnetium Compounds ; Paclitaxel ; therapeutic use ; Radiopharmaceuticals

OBJECTIVETo improve the X-ray diagnosis of cervical spondylosis of vertebral artery type (VCS).

METHODSA blinded design research. The X-ray signs both 60 patients with VCS and 60 patients with cervical spondylotic radiculopathy were collected from January 2011 to November 2012. There were 36 males and 84 females, aged from 25 to 65 years old with an average of (48.4 ± 12.3) years old. Cervical curvature, atlanto-occipital joint angle, atlanto-axial joint angle, C2/C3 joint angle and lower cervical instability condition and segmental distribution were measured and recorded by X-rays. These data were analyzed and compared between the two groups after unblended. Combined with clinical manifestations,the X-ray imaging features of VCS were further analyzed.

RESULTSThere was significant difference in cervical curvature between two groups in anteflexion X-ray films (P < 0.05). There was significant difference in extension degree of atlanto-occipital joint angle between two groups (P < 0.01). There was significant difference in atlanto-axial joint angle between two groups in lateral X-ray films (P< 0.05). There was significant.difference in anteflexion degree of atlanto-axial joint angle between two groups (P < 0.05). There was no significant difference in C2/C3 joint angle between two groups. There was no significant difference in the lower cervical instability condition and segmental distribution between two groups. In VCS group, the mild and moderate dizziness was main symptom, flexion and extension activities of neck was most common cause in the dizziness; and always accompanied with headache; tenderness mostly concentrated in the upper cervical area.

CONCLUSIONBoth X-ray signs and clinical manifestations can prompt the abnormalities of the upper cervical structure or function in patients with VCS. Anteflexion activities of neck observed by functional position of X-ray films should be emphasized in diagnosis of VCS.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Radiculopathy ; diagnostic imaging ; Radiography ; Spondylosis ; diagnostic imaging ; Vertebral Artery ; X-Rays

OBJECTIVETo observe the effects of mesenteric lymph duct (MLD) ligation on erythrocyte rheology in acute hemorrhagic rats.

METHODSTwenty male Wistar rats were randomly divided into hemorrhage group and ligation group (n = 10). Blood (one fourth of body whole blood volume) was withdrawn through right common carotid arteries after rats were anesthetized. In ligation group, the MLD was ligated after hemorrhage, and only threading under the MLD in hemorrhage group. The survival situation at 24 h was recorded. After 24 h, survival rats were anesthetized again, blood sample was withdrawn through left common carotid artery rapidly. And the erythrocyte sedimentation rate (ESR), electrophoresis of erythrocytes, hematocrit (Hct) were determined in blood samples of before and after hemorrhage, the erythrocytes aggregation and deformability indices were calculated.

RESULTSIt showed that the ligation group survival (9 rats alive) was slightly better than that in hemorrhage group (6 rats alive). The results of erythrocyte rheology indices showed that the ESR, K value of equation, K value of emendation and electrophoresis time in hemorrhage group and ligation group were higher or longer than those before hemorrhage, the erythrocyte deformability was reduced significantly, respectively. And the erythrocytes aggregation index in hemorrhage group was increased, the electrophoresis length and migration of erythrocyte in hemorrhage group were lower than those before hemorrhage, respectively. But compared with hemorrhage group, the ESR, K value of equation, K value of emendation, erythrocytes aggregation index and electrophoresis time in ligation group were lower, the electrophoresis lenght, migration and deformability of erythrocyte were increased significantly.

CONCLUSIONThe results indicate that the higher erythrocyte aggregation ability, lower electrophoresis function and deformability are caused by acute hemorrhage in rats, and the MLD ligation can improve the abnormal erythrocyte rheology.

Animals ; Disease Models, Animal ; Erythrocyte Deformability ; Erythrocytes ; pathology ; Hemorrhage ; surgery ; Ligation ; Lymphatic Vessels ; surgery ; Male ; Mesentery ; surgery ; Rats ; Rats, Wistar ; Rheology ; Shock, Hemorrhagic ; surgery

AIMTo observe the effect of mesenteric lymph duct ligation on free radical and inflammatory mediator of myocardium with severe hemorrhagic shock in rats at different period, and explore the effect of intestinal lymphatic pathway on myocardium injury pathogenesis in shock rats.

METHODS78 male Wistar rats were divided into the sham group, shock group and ligation group. The model of serious hemorrhagic shock was established in shock group, ligation group, and mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after resuscitate. All rats were executed and taken out heart making for homogenate of 10 percent to determine the MDA, SOD, tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), myeloperoxidase (MPO), NO and NOS at after shock 90 min, after transfusion and resuscitate 0 h, 1 h, 3 h, 6 h, 12 h and 24 h etc. different times, and the expression of inducible nitric oxide synthase (iNOS) mRNA in myocardium was detected by RT-PCR.

RESULTSThe contents of MDA, TNFalpha, IL-6, MPO, NO, NOS and iNOS expression in myocardium of shock group were rising after transfusion and resuscitate, and that was higher level at 3 h to 12 h, and that was significantly higher than sham group, the activity of SOD was significantly lower than sham group. The contents of MDA, TNFalpha, IL-6, MPO, NO, NOS and iNOS expression in myocardium of ligation group were significantly lower than that of shock group at sameness points, and the SOD activity was higher.

CONCLUSIONThe mesenteric lymph duct ligation and blocking mesenteric lymph could reduce the PMN detaining, decrease the discharging of TNFa and IL-6, reduce the NO and expression of iNOS mRNA, and reduce the releasing of free radical and consuming of SOD.

Animals ; Free Radicals ; metabolism ; Inflammation Mediators ; metabolism ; Interleukin-6 ; metabolism ; Lymphatic Vessels ; metabolism ; Male ; Mesentery ; metabolism ; Myocardium ; metabolism ; pathology ; Neutrophils ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Peroxidase ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; metabolism ; pathology ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effects of a new anticoagulant, annexin V derivative (AND) on anticoagulation and antithrombosis.

METHODSHigh and low doses of AND were given to rabbits (groups 1 and 2 respectively) by intravenous (iv) bolus injections followed by half the respective AND doses by iv infusion over 2 hours. Control groups were iv given heparin (group 3) and saline (group 4) of the same volume and procedure as that in group 1 and 2. Blood cell count, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen level were examined before and 15, 30 and 60 min after iv bolus and 2 hours after the end of iv infusion. A 3.0 mm x 15 mm balloon was put into femoral artery to induce endothelial denudation 15 min after IV bolus and the blood pressure of femoral artery was monitored until the pulse pressure recorded 0 mm Hg when the vessel was occluded completely by a thrombus. The femoral arteries were collected and the thrombi were stripped off for measuring their lengths, wet and dry weights.

RESULTSAnticoagulation parameters: APTT at 15 min after iv bolus in AND group was significantly longer than that in group 4 (P < 0.05) but shorter than that in group 3 (P < 0.05); APTT and TT in group 3 were significantly longer than those in groups 1, 2 and 4. Fibrinogen: 0.70 mg/kg AND may decrease fibrinogen. Antithrombosis values: the wet and dry weights in AND groups were significantly lighter than those in group 3 and 4 (P < 0.05). The dry weight in high-dose AND group was remarkably lighter than that in low-dose group (P = 0.029). The length of thrombus in low-dose AND group was remarkably shorter than that in group 4 (P = 0.013), but not for group 3 (P > 0.05). It was remarkably shorter in high-dose AND group than in both group 3 (P < 0.001) and 4 (P = 0.015). The time when pulse pressure equaled to 0 was longer in AND group than in group 4 (P < 0.05), but not in 3.

CONCLUSIONAND is an effective anticoagulant and antithrombosis agent, the highest anticoagulation effect occurs at 15 min after IV bolus. Its anticoagulation effect is not more potent than that of standard heparin, while antithrombosis capacity is more effective. AND in treating thrombosis clinically might be promising.

Animals ; Annexin A5 ; administration & dosage ; pharmacology ; Anticoagulants ; administration & dosage ; pharmacology ; Blood Coagulation ; drug effects ; Disease Models, Animal ; Fibrinogen ; analysis ; Humans ; Injections, Intravenous ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Rabbits ; Random Allocation ; Thrombin Time ; Thrombosis ; prevention & control

OBJECTIVETo prepare a drug-loading film using chitosan and carboxymethyl chitosan as the carrier materials for delivering matrine to oral ulcers.

METHODSMatrine-loading films using chitosan or carboxymethyl chitosan as the carrier materials were prepared by solution casting method and orthogonal experiment at room temperature. The mechanical properties, surface morphology and drug-loading capacity of the drug-loading film were characterized using tensile test, scanning electron microscopy (SEM), swelling test and in vitro drug release test.

RESULTSWhen the molecular weight of chitosan was 650 000 and the mass ratio of chitosan/glycerol was 1:1.4, the prepared film had the maximum mechanical strength and tensile modulus reaching 0.7875 MPa. SEM observation showed that matrine aggregated at the bottom of the drug-loading film with an asymmetrical distribution. The in vitro drug release test showed that the film had a high drug-loading capacity and a sustained drug release property. The duration of drug release from the drug-loading film was prolonged as the molecular weight of chitosan increased, reaching 23 h when the molecular weight of chitosan was 650 000. The duration of drug release was further increased to 108 h when the bottom of the drug-loading film was coated with a layer of 1% carboxymethyl chitosan.

CONCLUSIONThe matrix materials of the drug-loading film are natural, green, nontoxic and biodegradable, and the preparation of the film is simple without using large quantities of organic solvents. The novel drug-loading film can obviously prolong the duration of drugs release for better local drug delivery to oral ulcers in a sustained manner.

Alkaloids ; chemistry ; Chitosan ; analogs & derivatives ; chemistry ; Delayed-Action Preparations ; Drug Delivery Systems ; Drug Liberation ; Glycerol ; chemistry ; Microscopy, Electron, Scanning ; Quinolizines ; chemistry