Biomedical subjects

Tao Liu

Publications and source records attributed to Tao Liu.

At least 109 records · Page 6Linked to original sources

PubMed2005

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region.

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P(vgb) of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P(vgb) was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P(vgb) provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P(vgb), while a construct containing P(vgb), the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P(vgb), vgb operon) or pKVpPV (P(vgb), vgb operon, par) slightly attenuated (approximately 30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P(vgb), egfp) or pKVpP-E (P(vgb), par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P(vgb), vgb operon, par, tod) or pKVpP-T (P(vgb), par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.

Bacterial Proteins↗

PubMed2005

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

Amino Acid Sequence↗

PubMed2005

Molecular cloning, expression and characterization of hyoscyamine 6beta-hydroxylase from hairy roots of Anisodus tanguticus.

Anisodus tanguticus, one of the indigenous Chinese ethnological medicinal plants of the Solanaceae, produces anticholinergic alkaloids such as hyoscyamine, 6 beta-hydroxyhyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase (H6H), a key enzyme in the biosynthetic pathway of scopolamine, catalyzes the hydroxylation of hyoscyamine and epoxide formation from 6 beta-hydroxyhyoscyamine to generate scopolamine. A full-length cDNA of H6H has been isolated from A. tanguticus hairy roots. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence shows a typical motif of 2-oxoglutarate-dependent dioxygenase. The A. tanguticus H6H was expressed in Escherichia coli and purified for enzyme function analysis. This study characterized the recombinant AtH6H and showed it could generate scopolamine from hyoscyamine.

Amino Acid Sequence↗

PubMed2005

Molecular cloning and expression of putrescine N-methyltransferase from the hairy roots of Anisodus tanguticus.

A full-length cDNA encoding putrescine N-methyltransferase (PMT) was isolated from the hairy roots of A. tanguticus. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1017 bp encoding 338 amino acids with high homology to other known PMTs. A. tanguticus PMT was expressed in Escherichia coli. The recombinant AtPMT was purified and exhibited S-adenosyl-methionine-dependent N-methyltransferase activity.

Amino Acid Sequence↗

PubMed2005

Preparation of adsorbents for the removal of endotoxin.

By using the agar beads as the support, L-lysine as the ligand, three kinds of absorbents with different spacers for removal of endotoxin were prepared. The adsorption capacity of three adsorbents was compared. Among them, Ag3, with L-lysine ligand and hexamethylendiamine as the spacer, was the best in adsorption capacity and was selected for hemoperfusion on rabbits. The results showed the level of endotoxin in endotoxemia rabbit after hemoperfusion was near normal level. The clearance percentage of endotoxin was 73.6%

Adsorption↗

PubMed2005

Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers.

Livers from obese donors often have fatty infiltrates and are more susceptible to ischemia-reperfusion injury and subsequent graft dysfunction. This often leads to the exclusion of organs from obese donors. We investigated whether ischemic preconditioning (IP, 10 min ischemia, 10 min reperfusion) preserves cellular metabolism in livers from obese Zucker rats during cold ischemia. Liver samples (-IP and +IP) were collected from obese and control lean rats at different time points of cold ischemia (CI) and analyzed by magnetic resonance spectroscopy (1H- and 31P-MRS) to assess whether IP improves hepatic cellular metabolism. IP significantly improved high energy metabolism in IP livers from obese rats when compared with obese controls during the first hours of CI. At 4 h of cold storage, obese IP livers were not different from control lean non-IP livers. The beneficial metabolic effect of IP on livers form obese rats, however, was absent at 8 h of reperfusion. In contrast, in livers from lean rats, IP resulted in improved high-energy metabolism during the entire observation period of 8 h. In a later part of the study, IP of liver grafts from obese rats before 4 h of cold storage improved recipient survival after graft transplantation. IP of liver grafts from obese rats before 4 h of CI increases 24-h survival of recipient animals from 25% to 88%.

Adenosine Triphosphate↗

PubMed2005

Role of Cdx-2 in insulin and proglucagon gene expression: a study using the RIN-1056A cell line with an inducible gene expression system.

Although the homeobox gene Cdx-2 was initially isolated from the pancreatic beta cell line HIT-T15, no examination of its role in regulating endogenous insulin gene expression has been reported. To explore further the role of Cdx-2 in regulating both insulin and proglucagon gene expression, we established an ecdysone-inducible Cdx-2 expression system. This report describes a study using the rat insulinoma cell line RIN-1056A, which abundantly expresses both insulin and proglucagon (glu), and relatively high amounts of endogenous Cdx-2. Following the introduction of the inducible Cdx-2 expression system into this cell line and the antibiotic selection procedure, we obtained novel cell lines that displayed dramatically reduced expression of endogenous Cdx-2, in the absence of the inducer. These novel cell lines did not express detectable amounts of glu mRNA or the glucagon hormone, while their insulin expression was not substantially affected. In the presence of the inducer, however, transfected Cdx-2 expression was dramatically increased, accompanied by stimulation of endogenous Cdx-2 expression. More importantly, activated Cdx-2 expression was accompanied by elevated insulin mRNA expression, and insulin synthesis. Cdx-2 bound to the insulin gene promoter enhancer elements, and stimulated the expression of a luciferase reporter gene driven by these enhancer elements. Furthermore, Cdx-2 and insulin gene expressions in the wild-type RIN-1056A cells were stimulated by forskolin treatment, and forskolin-mediated activation on insulin gene expression was attenuated in the absence of Cdx-2. We suggest that Cdx-2 may mediate the second messenger cAMP in regulating insulin gene transcription.

Animals↗

PubMed2005

[A study of interferon-gamma induced airway mucous cell apoptosis and its mechanisms].

OBJECTIVE: To investigate the effect of interferon-gamma (IFN-gamma) on airway mucous cells and its mechanisms. METHODS: (1) Normal human bronchial epithelial cells (NHBEs) were cultured under specific conditions, and treated by IFN-gamma for 3 days. The cells were analyzed with fluorescein isothiocyanate (FITC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. (2) Twenty wild type C57BL/6J mice were immunized and divided into 4 groups, and treated with IFN-gamma (50 ng and 100 ng, respectively), interleukin-13 (IL-13, 5 microg) and saline by nostril instillation. The mice were sacrificed and the airway mucous cells were analyzed by morphometry and TUNEL assay. RESULTS: (1) IFN-gamma induced apoptosis in NHBEs, which showed condensed nuclei, nuclear and DNA fragmentaion, and were positive by TUNEL assay. Bax was upregulated and translocated from cell plasma to mitochondria under the treatment. (2) Airway mucous cell account in 100 ng IFN-gamma instillation immunized mice group (28 +/- 6 mucous cells/mm basal lamina) was significantly decreased as compared to that in saline (58 +/- 12) and IL-13 (59 +/- 6) instillation groups (all < 0.05). There was no difference among the IFN-gamma 50 ng (48 +/- 11), saline (58 +/- 12) and IL-13 (59 +/- 6) instillation groups (all P > 0.05). TUNEL assay was also positive in airway mucous cells from IFN-gamma instillation mice as compared to saline instillation mice. CONCLUSIONS: IFN-gamma leads to airway mucous cell apoptosis by Bax upregulating and translocation into mitochondria. This might be of significance in the new therapies of asthma.

Animals↗

PubMed2005

[Modified sanjiasan decoction in regulating intelligence state of patients with vascular dementia].

OBJECTIVE: To investigate the therapeutic effects of modified Sanjiasan decoction (MSD) on vascular dementia (VD). METHODS: Thirty-seven patients in the treated group were given MSD, one dosage each day, and 31 patients in the control group were administered orally Naofukang 0.8 g three times a day. The treatment course for both groups was three months. The indices as Hasegawa dementia scale (HDS), mini mental state examination (MMSE) and its subentries, intelligence quotient (IQ) and memory quotient (MQ) were examined. RESULTS: MSD could improve the scores of HDS, MMSE and its subentries (P<0.05 or P<0.01), ameliorate dementia state and enhance IQ (P<0.05) and MQ (P<0.01) in patients with VD. CONCLUSION: MSD has a certain effect on intelligence benefiting and dementia antagonizing.

Aged↗

PubMed2005

[Clone expression and purification of influenza A virus nucleoprotein gene].

OBJECTIVE: To clone the influenza A virus NP gene into expression vector and to purify the target protein, which was used to study the preparation of monoclonal antibody. METHODS: The RNA of influenza A virus was extracted and primers were designed according the NP gene sequence, then the NP gene of influenza A virus was expressed in E.coli DH5alpha and the NP protein was purified by affinity chromatography. RESULTS: The recombinant expression vector-pGEX-4T-2-NP was successfully constructed and relatively pure target protein was obtained. CONCLUSION: Through reasonably controlling the fermentation time, growth temperature and induction concentration, satisfactory soluble target product was obtained.

Base Sequence↗

PubMed2005

[Expression and assay in vitro of phage single chain antibody against nucleoprotein of influenza virus A type].

OBJECTIVE: To expression the phage single chain antibody against nucleoprotein of influenza virus A type in E. coli strain HB2151 by screening the positive clone from the phage antibody library against nucleoprotein, which will prepare for the construction of fast leak kit of Influenza virus A type. METHODS: The positive clone ratio in the phage antibody library was enriched by three-round continual solid panning, ELISA, SDS-PAGE and Western-blot methods were used to analyze the protein secreted into the supemants and periplasmic. Affinity chromatography was used to purified the protein expressed in periplasmic and the titer was also analyzed using ELISA method. RESULTS: 10 clones were screened from96 clones and among the 10, there were 3 stronger positive with OD450nm value of 0.469, 0.582 and 0.507, respectively. The phage single chain antibody against Influenza virus A type was primary expressed in periplasmic. The ELISA results were positive even if the single chain antibody purified by affinity chromatography was diluted 4096 folds. CONCLUSION: Using phage antibody library technique, phage single antibody against nucleoprotein of influenza virus A type was preliminary expressed in E. coli.

Antibodies, Viral↗

PubMed2005

[Detection of SARS-coronavirus in both human and animals by RT-PCR].

OBJECTIVE: To find the most suitable RT-PCR detection method for SARS-Coronavirus (SARS-CoV) detection in both human and animals, and to study the source of SARS virus by investigating the condition of virus carried by wild, domestic animals and animals sold in market. METHODS: 350 throat washes of confirmed, suspected and observed SARS cases were tested by TaqMan and molecular beacon fluorescence RT-PCR methods. 386 animals with 442 nasal-throat swabs, fecal swabs of animals and cell cultured were detected by TaqMan method and conventional RT-PCR method. RESULTS: 10 positive were detected from 41 SARS clinical confirmed cases (24.39%). The results of TaqMan and molecular beacon are basically coincident with the coincident rate of 88.89%. 18 cell cultures with CPE were detected and find 16 positive, among which, 14 were civet cats (87.5%). The detecting results of animal samples from Dongmen Market (Shenzhen) and other markets and farms in peripheral areas show that: the total positive rate of 4 kinds of wild animal (civet cat, raccoon dog, hog-badge and Chinese ferret-badge) is 39.02% , which has an significant difference (P < 0.01) compared with the positive rate of other animals as 0. The positive rate of PCR for nasal and fecal swabs from 109 major wild animals is 44.04%, but the positive rate of 145 wild live-animals is 0. CONCLUSION: TaqMan and molecular beacon PCR methods both can be used in SARS detection.The results could support the hypothesis that animals (especially civet cat) could carry SARS virus.

Animals↗

PubMed2005

[Modified molecular beacon-based dual real-time PCR for detection of SARS virus and its application].

OBJECTIVE: To develop the modified molecular beacon-based dual fluorescent PCR assays for detection of SARS virus. The assay was applied to the early clinic diagnosis & animal tracking. METHODS: On the basis of the obtained core sequence of open reading frame 1b of the coronavirus polymerase gene sequences, which was published in GenBank, using modified molecular beacon probe, artifical virus techinique and two different fragments amplification with different fluoresce, one set of primers and probe were designed. Then fluorescent PCR assays for specific and sensitive detection of the SARS virus was established, while the ELISA & the traditional method were used as control. 368 clinical specimens such as the throat swab, serum, feces, and urine from different cases, 52 cell cultures and 50 animal specimens were detected by the molecular beacon-based PCR. RESULTS: The sensitivity of real -time PCR was 10 - 100 copies/ml, there was no cross reaction to other respiratory viruses such as influenza virus etc. Of 368 specimens, 20 were positive by using molecular beacon-based fluorescence PCR. The positive rate of SARS case (10/47) were 21.27%, the positive rate of the throat swab of SARS cases (10/23) were 43.87% . Among 52 SARS cell cultures, 29 were positive. The positive rate of SARS cell cultures was 55.77% . Of 50 animal specimens, 23 were positive. The positive rate was 46%. Furthermore, SARS virus RNA was detected in feces and in serum during the acute phase. CONCLUSION: The molecular beacon-based PCR is sensitive and specific, it could be applied to the early diagnosis and animal tracking. This molecular beacon-based PCR kit is useful for the different units.

Animals↗

PubMed2005

[Study on the expression of HSP70 and HSP90beta in nasopharyngeal carcinoma and the clinical significance].

OBJECTIVE: To study the expression of HSP70 and HSP90beta and its clinical significance in human nasopharyngeal carcinoma. METHOD: Immunohistochemical method SP was used to detect the expression of HSP70 and HSP90beta in 50 cases and its clinical significance were studied. RESULT: The positive rate of HSP70 and HSP90beta were 72% and 56% respectively. Analysis of patients, survival demonstrated that the prognosis of NPC with HSP70 (-) and HSP90beta (-) expression were significantly better than the others. CONCLUSION: The overexpression of HSP70 and HSP90beta were probably concerned with the occurrence, development and prognosis of nasopharyngeal carcinoma.

Adolescent↗

PubMed2005

[Diagnosis and treatment of paraduodenal hernia: a report of 16 cases].

OBJECTIVE: To summarize the experience of diagnosis and treatment for paraduodenal hernia (PDH). METHODS: The clinical data of 16 cases with PDH surgically treated from 1962 to 2003 were analyzed retrospectively. RESULTS: The average age of the patients was 36 years old. There were 13 cases with left PDH (Landzerts hernia) and 3 cases with right PDH (Waldeyer's hernia). Ten patients usually had no symptoms, while acute abdominal pain occurred after full food in 3 case and after vigorous movement in 13 cases respectively. X-ray revealed complete or incomplete intestinal obstruction in 12 cases, while ultrasonography and CT revealed dilated cystic jejunal loop between the pancreas and the stomach in 6 cases. Fourteen cases (87.5%) were misdiagnosed before operation. The hospital stay ranged from 10 to 13 days. All patients were followed up from 2 to 4 years without recurrence. CONCLUSION: With high misdiagnostic rate, it is the key to perform exploratory operation for suspicious PDH.

Adolescent↗

PubMed2005

[Clinical experience of acupuncture and moxibustion for treatment of chronic cholecystitis].

OBJECTIVE: To sum up clinical experience of acupuncture and moxibustion for treatment of chronic cholecystitis and study on the factors of influencing the therapeutic effect of acupuncture and moxibustion treating chronic cholecystitis. METHODS: The factors of influencing the therapeutic effect were summarized from the indications of acupuncture and moxibustion, meridians, acupoint selection, selection of needling and moxibustion methods, time law of attack, treating the principal aspects and the secondary aspects of a disease, arrival of qi, combined treatment of acupuncture and medicine, lasting time and frequency of treatment, assessment of therapeutic effects, and so on. CONCLUSION: Acupuncture and moxibustion treatment of chronic cholecystitis fully reflects the characteristics and complex nature of the acup-moxibustion therapy. Only when concretely analyze practical condition of the patient and adopt acupuncture and moxibustion treatment, can good therapeutic effect be achieved.

Acupuncture↗

PubMed2005

[The protective effect of recombinant Chinese interferon-gamma in pulmonary injury in mice].

OBJECTIVE: To observe the protective role of recombinant Chinese interferon-gamma (INF-gamma) in pulmonary injury (PI) induced by bleomycin (BLM) in C57 mice. METHODS: Seventy-five C57 mice were randomly divided into a control group and groups treated with BLM, BLM + INF-gamma minimum dose (0.25 microg/d), BLM + INF-gamma medium dose (0.5 microg/d), and BLM + INF-gamma maximum dose (1.0 microg/d, with 15 mice each). PI was induced by BLM, and intervention with different doses of INF-gamma was carried out in the experiment groups, but no treatment was administered in the control mice. Measurement of pulmonary hydroxyproline (Hyp), image analysis of collagen I and III, and measurement of the ratio between lung alveoli and interstitial areas were performed. RESULTS: Lung Hyp content and collagen I and III deposition were increased as compared with the control after administration of BLM (0.78 +/- 0.08 vs 0.65 +/- 0.06, P < 0.01; 0.048 +/- 0.006 vs 0.004 +/- 0.001, P < 0.01). Hyp content and collagen I and III deposition were reduced in group INF-gamma maximum dose (0.67 +/- 0.08 vs 0.78 +/- 0.08, P < 0.05; 0.008 +/- 0.001 vs 0.048 +/- 0.006, P < 0.01). The ratio of lung collagen to lung tissue areas was increased with the increase of INF-gamma doses (1.78, 0.12, 0.67, 0.73, 1.65 respectively). CONCLUSION: INF-gamma is effective in alleviating BLM induced pulmonary injury in mice, possibly by inhibition of transformation of fibroblasts to myofibroblasts and collagen synthesis.

Animals↗

PubMed2005

[Clinical study on method of "tonifying the lung to check the liver" for treatment of chronic cholecystitis].

OBJECTIVE: To evaluate the significance of method of "tonifying the lung to check the liver" for treatment of chronic cholecystitis. METHODS: Forty cases were randomly divided into a treatment group and a control group. The treatment group were treated with acupuncture and moxibustion by the method of "tonifying the lung to check the liver", and the control group with routine moxibustion. RESULTS: After treatment of one day, pain improved significantly in the two groups (P < 0.001), with no significant difference between the two groups; and 15 days after treatment, the treatment group was superior to the control group in the improvement of pain. CONCLUSION: TCM syndrome differentiation has an important significance in clinical acupuncture and moxibustion, and the therapeutic principle "tonifying the lung to check the liver" in classical TCM theories is of scientific basis.

Acupuncture Therapy↗