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

OBJECTIVETo construct the recombinant plasmids expressing Skp2 short hairpin RNA (shRNA) by pRNAT-U6.1/Neo plasmid vector and observe the effects of RNAi-mediated Skp2 gene silencing on Tca8113 cells.

METHODSFive recombinant eukaryotic expression vectors were successfully constructed using pRNAT-U6.1/Neo plasmid vector separately. After they were transfected into Tca8113 cells with PEI, the interference effects no Skp2 and p27 were detected by RT-PCR and Western blot. The cell cycle of Tca8113 cells were tested by flow cytometry. The proliferation of Tca8113 cells were examined by MTT.

RESULTSIn Skp2shRNA-2 and Skp2shRNA-3 vectors, the expression of Skp2 protein of Tca8113 cells was down-regulated and p27 protein up-regulated (P < 0.01). The cell number during G1/G 0 phases increased 22% (P < 0.01) and during G(2)/M and S phases the number decreased 10% and 12% (P < 0.01). The proliferation of Tca8113 cells slowed down and the cells number decreased (P < 0.01).

CONCLUSIONSSkp2shRNA-2 and Skp2shRNA-3 vectors of shRNA for Skp2 were successfully constructed. They could influence expression of Skp2 and p27 gene. Skp2 may be a promising target of gene therapy on human tongue squamous cell carcinoma.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; genetics ; Humans ; Plasmids ; genetics ; RNA Interference ; S-Phase Kinase-Associated Proteins ; genetics ; metabolism ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection

OBJECTIVETo observe the effects of mesenteric lymph drainage on erythrocyte rheology and blood viscosity in hemorrhagic shock rats.

METHODSWistar rats were randomly divided into sham-shock group, shock group (establishing hemorrhagic shock model), drainage group (establishing hemorrhagic shock model plus drainaging shock mesenteric lymph from hypotension 1 h). At 3 h of hypotension or corresponding time, blood samples were harvested from the abdominal aorta for determining the erythrocytic parameters, erythrocyte electrophoresis, erythrocyte sedimentation rate (ESR) and blood viscosity, and the erythrocytes aggregation index and erythrocyte deformability index were calculated.

RESULTSCompared with the sham-shock group, the red cell contents, hematocrit (HCT), hemoglobin (Hb), mean corpuscular hemoglobin concentration (MCHC), erythrocyte electrophoretic rate and mobility, erythrocyte deformability index, whole blood viscosity, whole blood relative or reduced viscosity at low and high shear rates in shock group were observably lower, and mean corpuscular volume, electrophoretic time of erythrocyte, ESR, K value of equation and K value of emendation, erythrocytes aggregation index, plasma viscosity in shock group were increased markedly; the MCHC, erythrocyte electrophoretic rate and mobility, whole blood viscosity, whole blood relative viscosity at low and high shear rates in drainage group were reduced, and the red blood cell volume distribution width -SD (RDW-SD) was increased remarkably. At the same time, in drainage group, the HCT, RDW-SD, erythrocyte deformability index, whole blood viscosity and relative viscosity at low and high shear rates were higher, the ESR, K value of equation and K value of emendation, erythrocytes aggregation index, plasma viscosity were lower than that of shock group.

CONCLUSIONThe results indicate that the mesenteric lymph drainage could improve the erythrocyte rheological behavior, as a result, improve the hemorrheological properties in hemorrhagic shock rats.

Animals ; Blood Viscosity ; Drainage ; methods ; Erythrocyte Aggregation ; Erythrocyte Deformability ; Lymph ; Male ; Mesentery ; Rats ; Rats, Wistar ; Rheology ; Shock, Hemorrhagic ; blood ; therapy

OBJECTIVETo report a novel deafness-causing mutation c.465T>A, p.Y155X in connexin 26 (CX26) (also called gap junction protein beta-2, GJB2), and perform functional analysis of the mutated protein p.Y155X in Hela cells to explore the underlying mechanism on deafness.

METHODSMutations in CX26 gene of the proband in an autosomal recessive inherited deafness family were tested by direct DNA sequencing method. Mutant p.Y155X, which was found in the deafness family, and wild type CX26 (wtCX26), were directionally subcloned into the pEGFP-N1 plasmid to construct the recombinant fusion protein expression vector of CX26 p.Y155X-EGFP and wtCX26-EGFP, followed by transfecting into HeLa cells. The expression of the mutated and wild type proteins was analyzed using Western blot analysis. The intracellular localization of proteins and the formation of gap junction-like plaques at plasma membrane were observed under confocal microscope. Gap junction coupling was tested by calcein-AM dye transfer experiment.

RESULTSA novel nonsense mutation c.465T>A, p.Y155X in the CX26 gene was found in the autosomal recessive deafness family. The molecular weight of protein p.Y155X was smaller than that of wtCX26 in transiently expressed HeLa cells. The mutated protein failed to reach the cell surface to form gap junction plaques, and displayed cytoplasmic accumulation. Also, no calcein-AM dye was transferred from the donor cells to the recipient cells when both were transfected with CX26 p.Y155X. The wtCX26 protein localized at the cell membrane to form gap junction plaques with permeability to fluorescent dye calcein AM.

CONCLUSIONCX26 p.Y155X could not be targeted to the plasma membrane and there was no formation of gap junction channels between the adjacent cells. The mutation c.465T>A, p.Y155X in CX26 gene was responsible for the autosomal recessive hearing impairment in this family.

Amino Acid Sequence ; Child ; Codon, Nonsense ; genetics ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; Deafness ; genetics ; Female ; HeLa Cells ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Sequence Homology, Amino Acid