Biomedical subjects

G Du

Publications and source records attributed to G Du.

At least 19 recordsLinked to original sources

PubMed2006

A fast online hit verification method for the single ion hit system at GSI.

For a single ion hit facility built to irradiate specific targets inside biological cells, it is necessary to prove that the ions hit the selected targets reliably because the ion hits usually cannot be seen. That ability is traditionally tested either indirectly by aiming at pre-etched tracks in a nuclear track detector or directly by making the ion tracks inside cells visible using a stain coupled to special proteins produced in response to ion hits. However, both methods are time consuming and hits can be verified only after the experiment. This means that targeting errors in the experiment cannot be corrected during the experiment. Therefore, we have developed a fast online hit verification method that measures the targeting accuracy electronically with a spatial resolution of +/-1 microm before cell irradiation takes place.

Cell Culture Techniques↗

PubMed2005

Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH-stress strategy.

AIMS: To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. METHODS AND RESULTS: When C. utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1.5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5.5 to 1.2 at 24 h and maintained at 1.2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02-08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1.2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. CONCLUSIONS: Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197.3 mg l-1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2.11 to 2.67% (w/w), and the total GSH concentration could reach 737.1 mg l-1 and increased by 24.9%. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.

Candida↗

PubMed2004

Hand-assisted transperitoneal laparoscopic living donor nephrectomy.

OBJECTIVE: We introduced and evaluated the advantages and disadvantages of the hand-assisted transperitoneal laparoscopic technique for living donor nephrectomy. MATERIALS AND METHODS: In December 2001, we started using the technique of hand-assisted transperitoneal laparoscopic living donor nephrectomy (HLDN) in 10 cases. The procedure utilizes a hand-assisted device to increase safety and control of the laparoscopic technique. RESULTS: Only left nephrectomy was performed. The mean total operating and the warm ischemia times were 130 minutes and 3.0 minutes, respectively. Average lengths of renal artery and vein were 1.95 cm and 2.8 cm, respectively. There were no intraoperative or postoperative complications. CONCLUSIONS: HLDN is an easier procedure than the traditional laparoscopic living donor nephrectomy and can greatly mitigate the learning curve. HLDN has shortened warm ischemia time and operating time. It is also good for trocar placement, prevention of torsion of the kidney, control of potential bleeding at the final stage of vascular stapling, and kidney removal. Therefore, HLDN is a promising method for living donor nephrectomy.

Adult↗

PubMed2003

A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation.

Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 microM UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R2=0.97), UDP-galactose epimerase (R2=0.75) and UDP-galactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.

Corynebacterium↗

PubMed2001

A role for phospholipase D1 in neurotransmitter release.

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca(2+)-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

Acetylcholine↗

PubMed2001

Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

BACKGROUND: Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood. METHODS: Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor. These methods were compared with antibody selection directly from whole blood. The total yield of erythroblasts was determined for each method. RESULTS: Red cell lysis is not recommended because the lysis step cannot be well controlled. Density separation followed by antibody selection works well. However, a faster and simpler method, antibody selection directly from whole blood using Immunicon Ferrofluid and magnetic separators, works as well and has the potential to yield even more cells. CONCLUSIONS: Considering the need for a simple and quick method for selecting fetal cells from maternal blood, we suggest selection directly from whole blood.

Acetazolamide↗

PubMed2001

Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two-stage culture system.

Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1 g l(-1) at 0.21 h(-1) of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6 g l(-1) at 0.14 h(-1) of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43 g l(-1) h(-1) at a dilution rate of 0.12 h(-1), but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36 g g(-1) glucose at 0.075 h(-1) of dilution rate with relatively high PHB productivity of 1.23 g l(-1) h(-1) and PHB content of 72.1%, respectively.

Cupriavidus necator↗

PubMed2001

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells.

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.

Actins↗

PubMed2001

Expression of multidrug-associated protein, P-glycoprotein, P53 and Bcl-2 proteins in bladder cancer and clinical implication.

The expression of multidrug resistant proteins in bladder cancer and clinical implication was studied. Expression of multidrug-associated protein (MRP), P-glycoprotein (P-gp), P53 and Bcl-2 proteins were detected by using immunohistochemical method in 40 specimens of bladder transitional cell carcinoma. The results showed that the positive rate of MRP, P-gp, P53 and Bcl-2 was 52.5%, 57.5%, 47.5% and 62.5% respectively. The positive rate of MRP, P-gp, P53 and Bcl-2 in the grade I, II and III of tumors was 46.3%, 38.5%, 38.5%, 23.1%; 52.9%, 39.8%, 47.1%, 76.4%; 60.0%, 80.0%, 60.0%, 90.0% respectively. The positive rate of MRP, P-gp, P53 and Bcl-2 in 24 primary tumor specimens was 37.5%, 41.7%, 33.3%, 45.8% and that in 16 cases in recurrent specimens receiving chemotherapy 75.0%, 81.3%, 68.8%, 87.5% respectively. It was suggested the positive rate of MRP, P-gp, P53 and Bcl-2 was increased with the advance of tumor grade. The positive rate of four proteins in all recurrent cases was significantly increased (P < 0.05). The expression of MRP, P-gp, P53 and Bcl-2 proteins might be the important factors for chemotherapy failure.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

ASH2L: alternative splicing and downregulation during induced megakaryocytic differentiation of multipotential leukemia cell lines.

Abstract Drosophila ash2 is a member of the trxG gene super family, some human homologues of which are involved in hematopoiesis and leukemia. We report here the identification of the human homologue of Drosophila ash2 and its alternative splicing isoform, ASH2L1 and ASH2L2. ASH2L proteins are 60% homologous to Drosophila ash2. ASH2L also has a zinc finger motif (C2C2) although it is not identical to that in ASH2. Expression profile analysis showed that the amount of ASH2L transcripts is extremely high in fetal liver, testis, and leukemia cell lines with erythroid and megakaryocytic potential such as K562, Hel, and Dami. We treated these cells with differentiation inducers phorbol ester and hemin. We found that ASH2L is downregulated rapidly and dramatically in K562, Hel, and Dami cells during phorbol ester induced differentiation with megakaryocytic features. However, its expression is maintained at a high level during erythroid differentiation of K562 cells induced with hemin. These results suggest that ASH2L plays a role in hematopoiesis and is associated with some special kinds of leukemia.

Alternative Splicing↗

PubMed2001

[The expression of HCV RNA polymerase in E.coli and the study of its solubility and antigenicity].

OBJECTIVE: To express HCV RNA polymerase and study its soluble condition and antigenicity. METHODS: We constructed expression vectors pQE-5B-Fl and pQE-5B-C21 and expressed them in E.coli (M15). We analyzed their solubility in different conditions and purified soluble pQE-5B-C21 protein by Ni-NTA column, then detected its antigenicity by ELISA and western blot. RESULTS: We obtained the purified soluble pQE-5B-C21 protein in the induction conditions of 18 degrees C and the protein was proved to be of good antigenicity by ELISA and western blot. CONCLUSIONS: The RNA polymerase of HCV expressed in E.coli has good solubility and antigenicity.

Blotting, Western↗

PubMed2001

[Brain-stem cavernous hemangioma: surgical indications and approaches].

OBJECTIVE: To evaluate the surgical indications and prognosis for cavernous hemangiomas (CHs) in the brain-stem. METHODS: From 1992 to 2000, 163 patients with intracranial CHs were operated on in our department. Among them, 14 patients (8 women and 6 men; mean age 42 years) had 15 CHs in the pons (12), mesencephalon (1) and medulla oblongata (1). Surgical indications for CHs are: progressive focal neurological deficits; hemorrhage; near the surface of the brainstem. Transvemian approach with midline suboccipital craniotomy was used for the lesions in the pons and medulla, near the floor of the IV cerebral ventricle. Transpetrosal approach was used for the lesions in the anterior part of the pons, and infratentorial supracerebellar approach for those in the mesencephalic case. RESULTS: CHs were totally removed from all the 15 lesions. Neurological deficits were improved in 10 patients after operation. Two patients had transient worsening of neurological conditions, which returned to the preoperative status during follow-up. The mesencephalic case, however had neurological condition deteriorated and was homebound after surgery. Pathologically, an oligodendroglioma was seen with CH in this case. CONCLUSIONS: If surgically indicated, CHs involving the brain-stem can be resected. Minimal invasiveness techniques and careful operative planning may contribute to the treatment of CHs involving the brain-stem.

Adolescent↗

PubMed2001

Management of hemangiopericytomas in the central nervous system.

OBJECTIVE: To elucidate the clinical features and treatment of Hemangiopericytomas (HPCs) in the central nervous system. METHODS: Twenty-six HPC operations performed at Huashan Hospital from January 1993 to June 1999 were analyzed retrospectively, and a review of relevant literatures was done. RESULTS: Among the 26 cases, total tumor removal was achieved in 24 cases, and subtotal removal in 2 cases. Postoperatively, neurological symptoms were improved or unchanged in 21 cases, worsened in 4 cases, and pneumonia caused death in 1 case. Eighteen patients received postoperative radiotherapy. Eighteen patients were followed up from 1 month to 58 months (average, 22 months). During the follow-up period, 2 patients presented one or more distant metastasis, and one patient died of recurrence. CONCLUSION: Surgical management is the best choice for treatment of patients with HPCs. Postoperative radiotherapy can postpone the risk of tumor recurrence.

Adolescent↗

PubMed2001

[Identification of the genome of a picornavirus isolate using Random-PCR].

OBJECTIVE: To identify the genome of an enterovirus isolate (XJ90). METHODS: The double-stranded cDNA of isolated virus was synthesized with anchored-random primer and was amplified with anchor primer; the PCR products were cloned into pGEM-T vector for sequencing and analysis. RESULTS: Analysis on agarose gel electrophoresis of the Random-PCR products showed a typical smear; 11 randomly selected clones were all homologous to the members of enteroviruses and the homology rate over 80%; the 11 clones were distributed evenly along the whole genome and they assembled 4 contigs totally 2 261 bp long which covered 30% of the genome. The homology of the fourth contig and coxsackie virus B6 was 97.3% at amino acids level. The virus gene was specifically amplified with primers derived from the sequenced clones. CONCLUSIONS: The picornavirus isolate XJ90 is a member of enteroviruses.

Amino Acid Sequence↗

PubMed2000

Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase C gamma and protein kinase C alpha signalling cascade.

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.

Cell Line↗

PubMed2000

Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner.

Myocardial phospholipase D (PLD) has been implicated in the regulation of Ca(2+) mobilization and contractile performance in the heart. However, the molecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot analysis, we found that PLD2 is the major myocardial PLD and that it localizes primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac protein was detected using a protein overlay assay employing purified PLD2 and then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct association between PLD2 and alpha-actinin was confirmed using an in vitro binding assay and localized to PLD2's N-terminal 185 amino acids. Purified alpha-actinin potently inhibits PLD2 activity (IC(50) = 80 nm) in an interaction-dependent and ADP-ribosylation factor-reversible manner. Finally, alpha-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is reciprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reorganization of the actin cytoskeleton.

Actinin↗

PubMed2000

Correlative expression of glutathione S-transferase-pi and multidrug resistance associated protein in bladder transitional cell carcinoma.

In order to elucidate the mechanisms of multidrug resistance (MDR) in bladder cancer, the expression of glutathione S-transferase-pi (GST-pi) and multidrug resistance associated protein (MRP) in tissue samples resected from 44 patients and 6 normal bladder mucosa as control was detected by using immunohistochemical method, and the results were analyzed by computer-assisted image analyzing system (IAS) to achieve semi-quantitative data. In addition, correlation between the expression of both factors was studied. The results showed that the positive expression rate of GST-pi and MRP in bladder cancer was 72.7% (32/44) and 68.2% (30/44) respectively, significantly higher than those in normal bladder mucosa, being 16.7% and 33.3% respectively. The rate of GST-pi positive staining was increased correspondingly with tumor grade and stage elevated, being higher in recurrent tumors treated by chemotherapy, but not significantly (P > 0.05). There was no significant differences between the expression of MRP and tumors' behaviors and clinical characters. However, the results demonstrated that the correlation between the expression of both resistant factors was very evident (r = 0.695, P < 0.0025). It was suggested that the activation of GST-pi and MRP might occur during malignant transformation of normal mucosa, but tumors' differentiation and progression could not be the unique factors that influenced both overexpression. Chemotherapy might be another important reason. The correlation of both indicated that there was a common mechanism regulating their expression probably, which made them play a pivotal role in chemotherapy drug resistance of bladder cancers.

Adult↗

PubMed2000

Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling.

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.

ADP-Ribosylation Factor 1↗