Animals ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; genetics ; Gene Expression ; Genetic Engineering ; methods ; Mice ; Molecular Sequence Data ; Mutation ; Promoter Regions, Genetic ; genetics ; RNA Polymerase II ; genetics ; RNA, Guide ; genetics

OBJECTIVENeurogenic pulmonary edema (NPE ) was indicative of poor prognosis in the epidemic of enterovirus 71 infections. The pathogenesis of NPE remains poorly understood. The objectives of this experimental study were to explore whether RAS is activated during NPE in rabbit models induced by fibrin and the effects of an angiotensin converting enzyme inhibitor (enalaprilat) on NPE.

METHODNPE models were induced by intracisternal injection of fibrinogen and thrombin. According to random number table method, 18 healthy adult New Zealand rabbits were assigned to three groups (with 6 in each) : normal control group (Con group), NPE group and enalaprilat treated (Ena) group. After establishment of NPE models, rabbits in Ena group were given intravenous enalaprilat 0.5 mg/kg. Expression of ACE,ACE2,AT1R mRNA of the lung tissue were evaluated by real-time polymerise chain reaction; and Ang II of the lung tissue was determined by enzyme linked immunosorbent assay ( ELISA ). Meanwhile, histopathological lung injury scores were evaluated.

RESULTACE mRNA expression level in NPE group ( 17.2 ± 3.3) appeared an increasing trend in contrast to Con group ( 12.6 ± 5.2 ) and Ena group ( 11.5 ± 2.4, both P > 0.05 ). Compared with Con group (81 ± 22 ), ACE2 mRNA expression levels of NPE group ( 52 ± 6 ) and Ena group ( 45 ± 13 ) both decreased ( both P < 0.05 ) . ACE mRNA/ACE2 mRNA expression levels of NPE group ( 0.33 ± 0.06 ) and Ena group ( 0.26 ± 0.04 ) were higher than those of Con group ( 0.16 ± 0.05, both P < 0.05 ), as well as the ratio of Ena group decreased compared with untreated NPE group ( 0.26 ± 0.04 vs. 0.33 ± 0.06, P < 0.05 ) . There were no statistically significant differences in expression of AT1 mRNA of the lung tissue among three groups, but Ena group ( 4.8 ± 1.1) in contrast to NPE group ( 6.7 ± 1.3) has no significant difference (P > 0.05). Lung AngII level of NPE group [(540 ± 147) pg/ml] was significantly higher than that of Con group [(253 ± 37 ) pg/ml] and Ena group [(309 ± 35 ) pg/ml, both P < 0.05 ]. Gross pathologic examination showed that pink foamy edema fluid appeared in the tracheal tubes in NPE group, but spontaneously appeared in neither Con group nor Ena group; and the level of pulmonary subpleural bleeding in Con group, 12 graded 0; in NPE group, 2 graded II, 10 graded III; in Ena group, 2 graded, 8 grade II, 2 grade III. The histopathologic lung injury scores in Ena group was decreased in contrast to NPE group (1.36 ± 0.26 vs.2.32 ± 0.49, P < 0.05) and mainly for the improvement of alveolar overdistension and interstitial edema.

CONCLUSIONThe present study showed that when NPE occurs, a high lung AngII concentration was associated with an imbalance between ACE mRNA to ACE2 mRNA expression level. Activated local RAS in lung tissue resulted in lung injury. Enalaprilat treatment may attenuate lung injury by interventing local RAS in lung tissue with decreased ratio of ACE mRNA to ACE2 mRNA and lung AngII concentration. The result will be significant for the angiotensin converting enzyme inhibitor used in the theatment of NPE.

Angiotensin II ; genetics ; metabolism ; Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Disease Models, Animal ; Enalaprilat ; pharmacology ; Female ; Fibrinogen ; pharmacology ; Gene Expression Regulation, Enzymologic ; Lung ; metabolism ; pathology ; Male ; Peptidyl-Dipeptidase A ; genetics ; metabolism ; Pulmonary Edema ; chemically induced ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Rabbits ; Random Allocation ; Real-Time Polymerase Chain Reaction

OBJECTIVETo construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.

METHODSThe fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.

RESULTSThe human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.

CONCLUSIONFusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.

Antigens, Surface ; immunology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; immunology ; Glutamate Carboxypeptidase II ; immunology ; Humans ; Male ; Polymerase Chain Reaction ; RNA, Small Interfering ; administration & dosage ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; immunology

Molecular mechanisms governing peritonitis caused by the presence of aseptic gauze have remained unclear. To identify the genes involved, sterile gauze-exposed omentum was collected at 0, 6, 12, 24, and 48 h intervals, and analyzed by differential display RT(reverse transcription)-PCR. Among over 1,200 bands, 230 bands were found differentially expressed. These bands represented the fragment sizes of approximately 200 to 1,500 bp. The eight fragments were expressed differentially in the treatment group but not in the control. The sequences of two bands were similar to those of genes associated with the inflammatory process and a band was related to repair and regeneration process. Another one was related with spermatogonia and the rest four were unknown. Additionally, amplicons corresponding to the full-length sequences of two inflammatory gene fragments were synthesized by rapid amplification of cDNA end PCR. One showed 99% similarity to the major histocompatibility complex class II dog leukocyte antigen-DR beta chain and the other was canis familiaris proteasome beta type 3. Results of the present study suggested that sterile gauze induced the differential expression of genes in the omentum involved in inflammation and healing process.
Animals ; *Bandages ; Base Sequence ; DNA, Complementary/analysis ; Dogs/*genetics/metabolism ; Gene Expression Profiling/veterinary ; Gene Expression Regulation ; Histocompatibility Antigens Class II/*genetics/metabolism ; Molecular Sequence Data ; Omentum/*metabolism ; Proteasome Endopeptidase Complex/*genetics/metabolism ; RNA, Messenger/analysis ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; *Wound Healing

BACKGROUNDThe relationship between apoptosis and tumors is a major focus in cancer research. RNA interference (RNAi) technology has emerged as a very potent tool to generate cellular knockdown phenotypes of a desired gene. The aim of this study was to explore the effect of siRNA specific to the protein casein kinase 2α (CK2α) on apoptosis of laryngeal carcinoma cells and to explore possible mechanisms.

METHODSAn siRNA expression plasmid specific to CK2α, psiRNA-hH1neo-CK2α, and a non-specific siRNA expression plasmid, psiRNA-hH1neo-cont, were constructed and transfected into Hep-2 cells by a lipofectamine method. The mRNA and protein levels of CK2α in transfected cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining methods. The morphological changes to Hep-2 cells were observed under transmission electron microscopy (TEM). The levels of Bcl-2 and Bax proteins were measured by Western blotting analysis.

RESULTSLevels of CK2α mRNA and protein were significantly decreased in the psiRNA-hH1neo-CK2α group compared to the other groups (P < 0.05). The apoptotic rate of the psiRNA-hH1neo-CK2α transfected group was significantly higher compared to that in the untransfected group and the siRNA-hH1neo-cont transfected group (25.66% ± 0.83%, 3.66% ± 0.43%, and 5.18% ± 0.22%) (P < 0.05). Compared with the untransfected group and the siRNA-hH1neo-cont transfected group, the psiRNA-hH1neo-CK2α transfected group presented with classical ultrastructural features of apoptosis, such as karyopyknosis, chromatic agglutination adjacent to the nuclear membrane, and apoptotic bodies. Compared with the other two groups, the level of Bcl-2 protein in the psiRNA-hH1neo-CK2α transfected group was decreased (0.20 ± 0.09 vs. 0.72 ± 0.16, 0.56 ± 0.11, P < 0.01), while the Bax protein level was increased (0.81 ± 0.17 vs. 0.26 ± 0.12, 0.33 ± 0.17, P < 0.01) and the ratio of Bcl-2 to Bax was decreased (0.25 ± 0.05 vs. 2.76 ± 0.21, 1.70 ± 0.22, P < 0.01).

CONCLUSIONSThe siRNA expression plasmid specific to CK2α could suppress CK2α expression and induce the apoptosis of laryngeal carcinoma cells. This is possibly by decreasing the Bcl-2/Bax ratio. CK2α may provide a potential therapeutic target against human laryngeal carcinoma.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Casein Kinase II ; genetics ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; bcl-2-Associated X Protein ; genetics ; metabolism

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.
Adult ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; CpG Islands ; genetics ; DNA Methylation ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Liver ; metabolism ; pathology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Middle Aged ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Transcription, Genetic

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.
Cell Line ; Cell Nucleus ; metabolism ; Cell Survival ; drug effects ; Gene Expression Profiling ; HeLa Cells ; Humans ; RNA Interference ; RNA Polymerase II ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Small Interfering ; pharmacology

Blood-borne angiotensin-II (Ang-II) has profound effects in the brain. We tested the hypothesis that Ang-II-dependent hypertension involves differential Ang-II type I (AT1) receptors expression in the subfornical organ (SFO) and the rostral ventrolateral medulla (RVLM). Male Wistar rats were implanted with 14-day osmotic minipump filled with Ang-II (150 ng/kg/min) or saline. AT1 receptor mRNA levels were detected in the SFO and RVLM by reverse transcription-polymerase chain reaction (RT-PCR). Ang-II caused hypertension (134 +/- 10 mmHg vs. 98 +/- 9 mmHg, n = 9, p < 0.05). RT-PCR revealed that Ang-II infusion induced increased AT1 receptor mRNA levels in RVLM and decreased in SFO. Our data suggest that Ang-II-induced hypertension involves differential expression of brain AT1 receptors.
Angiotensin II/*metabolism ; Animals ; Hypertension/chemically induced/*metabolism ; Male ; Medulla Oblongata/*metabolism ; RNA, Messenger/genetics ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 1/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Subfornical Organ/*metabolism

We investigated whether the detection of prostate specific membrane antigen (PSMA) in blood preoperatively has predictive value for biochemical recurrence (BCR) after radical prostatectomy in patients with prostate cancer. All 134 patients scheduled to receive radical prostatectomy for prostate cancer were prospectively enrolled. The authors used nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect PSMA mRNA-bearing cells in peripheral blood, and analyzed the ability of PSMA mRNA positivity to predict BCR after surgery. PSMA-mRNA was detected in 24 (17.9%) patients by RT-PCR. Over a median follow-up of 20 months (range, 3 to 46 months), BCR developed in 15 patients (11.2%) and median time to BCR was 7 months (range, 3 to 25 months). Kaplan-Meier analysis revealed a significant difference between those positive or negative for PSMA in terms of recurrence-free actuarial probability (log rank P=0.0039). Multivariate analysis showed that positivity for PSMA mRNA (HR: 3.697, 95% CI 1.285-10.634, P=0.015) and a biopsy Gleason score of > or =7 (HR: 4.500, 95% CI 1.419-14.274, P=0.011) were independent preoperative predictors of BCR. The presence of PSMA mRNA in peripheral blood can be used to predict BCR after radical prostatectomy.
Aged ; Antigens, Surface/*blood/genetics ; Glutamate Carboxypeptidase II/*blood/genetics ; Humans ; Male ; Middle Aged ; Neoplasm Recurrence, Local/blood/*diagnosis ; Predictive Value of Tests ; *Prostatectomy ; Prostatic Neoplasms/blood/*diagnosis/surgery ; RNA, Messenger/*blood ; Reverse Transcriptase Polymerase Chain Reaction ; Severity of Illness Index ; Time Factors

BACKGROUNDGenetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed.

METHODSThe silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used.

RESULTSThe Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05).

CONCLUSIONSilencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.

Animals ; Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Blotting, Western ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Female ; Flow Cytometry ; Gene Silencing ; physiology ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Mice ; Neoplasms ; immunology ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction