Biomedical subjects

J Guan

Publications and source records attributed to J Guan.

At least 37 records · Page 2Linked to original sources

PubMed2003

Effect of floc size and structure on biosolids capillary suction time.

Both size and structure of wastewater solids (biosolids) have been hypothesised to have an impact on the dewaterability of these solids yet very little data exists to validate this hypothesis or to elucidate the relative effects of size and structure. We have recently undertaken studies in which the size and structure of activated sludge flocs were altered in a controlled manner and the dewaterability of resultant flocs examined using the well established capillary suction time (CST) test. A small angle light scattering method was applied for the determination of activated sludge floc size and structure. The results obtained in this work show that floc structure is a major determinant of capillary suction time. In some instances, flocs have almost double the median (D(4,3)) size but possess similar fractal dimensions and similar CST values.

Bacteria↗

PubMed2002

Multilevel structure of sludge flocs.

In this work, the structure of two kaolin sludges and a waste activated sludge are investigated using both light-scattering and free-settling methods. Fractal dimensions estimated by the light-scattering and free-settling techniques (D(S) and D(F) respectively) differ significantly and support the hypothesis that naturally occurring aggregates possess a multilevel structure. A two-level floc structural model comprised of (i) a primary floc (of fractal dimension D(S)) consisting of primary particles and (ii) a secondary floc (of fractal dimension D(F)) consisting of the microflocs is proposed to interpret the experimental findings. The structural changes of sludge flocs before and after cationic flocculation are interpreted using the proposed two-level model.

Journal Article↗

PubMed2001

Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.

Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.

Androstadienes↗

PubMed2001

Near-infrared emissions in single-bubble and multibubble sonoluminescence.

Comparisons of light emissions from multibubble and single-bubble sonoluminescence in the near-infrared band extending from 800 to 1050 nm have been investigated. In argon-water mixtures, single-bubble and multibubble spectra are similar in appearance. In sodium chloride-water mixtures, the multibubble spectrum shows evidence of the 3d-3p sodium emission line, whereas the single-bubble spectrum shows no such emission. For single bubbles, the near-infrared emissions change linearly with driving pressure. No evidence of near-infrared emissions are observed below the visible luminescence threshold.

Journal Article↗

PubMed2001

Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole.

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Aminopeptidases↗

PubMed2001

Regulation of dna replication after heat shock by replication protein a-nucleolin interactions.

Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44 degrees C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60-90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20-30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPA-nucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.

DNA Replication↗

PubMed2001

Atp-binding cassette transporter ABC2/ABCA2 in the rat brain: a novel mammalian lysosome-associated membrane protein and a specific marker for oligodendrocytes but not for myelin sheaths.

We recently cloned a full-length cDNA of the rat ATP-binding cassette transporter 2 (ABC2, or ABCA2) protein, a member of the ABC1 (or ABCA) subfamily (-ABC1/ABCA1 is a causal gene for Tangier disease) and found it to be strongly expressed in the rat brain. In this study, we identified ABC2 as a lysosome-associated membrane protein that is being localized specifically in oligodendrocytes. The ABC2-immunolabeled cells were detected mainly in the white matter but were also scattered in gray matter throughout the whole brain. In addition, these cells were found to be colocalized with 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) immunoreactivity when the marker antibody for oligodendrocytes was used. However, no such colocalization was observed with markers for other kinds of glial cells. Unlike the CNP antibody, which also intensely stains myelin sheaths in the white matter, ABC2 immunoreactivity was detected only in the cell bodies of oligodendrocytes. At the ultrastructural level, ABC2 immunoreactivity was detected mostly around lysosome and partly in Golgi apparatus by electron microscopy. This was confirmed by immunocolocalization of ABC2 and lysosomal markers in a neuroblastoma cell line. Immunoblotting analysis of ABC2 from the whole brain and the ABC2-transfected cell line revealed bands at approximately 260 kDa. The result of in situ hybridization with a riboprobe for ABC2 matched the results obtained from immunostaining. These findings strongly suggest that ABC2 is a specific marker for oligodendrocytes but not for myelinsheaths and that it is as a novel mammalian lysosome-associated membrane protein involved in myelinization or other kinds of metabolism in the CNS.

ATP-Binding Cassette Transporters↗

PubMed2001

Reactivity of calix-tetrapyrrole SmII and SmIII complexes with acetylene: isolation of an "N-confused" calix-tetrapyrrole ring.

The nature of the substituents present on the calix-tetrapyrrole tetra-anion ligand [[R2C(C4H2N)]4]4- (R = [-(CH2)5-]0.5, Et) determines the type of reactivity of the corresponding SmII compounds with acetylene. With R = [-(CH2)5-]0.5, dehydrogenation occurred to yield the nearly colorless dinuclear diacetylide complex [[[[-(CH2)5-]4-calix-tetrapyrrole]SmIII]2(mu-C2Li4)].THF as the only detectable reaction product. Conversely, with R = Et, acetylene coupling in addition to dehydrogenation resulted in the formation of a dimeric butatrienediyl enolate derivative [[(Et8-calix-tetrapyrrole)SmIII[Li[Li(thf)]2(mu-OCH=CH2)]]2(mu,eta2,eta'2-HC=C=C=CH)]. Reaction of the trivalent hydride [(Et8-calix-tetrapyrrole)(thf)SmIII[(mu-H)[Li(thf)]]2 or of the terminally bonded methyl derivative [(Et8-calix-tetrapyrrole)(CH3)SmIII[[Li(thf)]2[Li(thf)2](mu3-Cl)]] with acetylene resulted in a mixture of the carbide [[(Et8-calix-tetrapyrrole)SmIII]2(mu-C2Li4)].Et2O with the dimerization product [[(Et8-calix-tetrapyrrole)SmIII[Li[Li(thf)]2(mu3-OCH=CH2)]]2-mu,eta2,eta'2-HC=C=C=CH)]. The same reaction also yielded a third product, a trivalent complex [[(Et8-calix-tetrapyrrole)SmIII[Li(thf)2]]2], in which the macrocycle was isomerized by shifting the ring attachment of one of the four pyrrole rings.

Journal Article↗

PubMed2001

Insulin-like growth factor-1 improves somatosensory function and reduces the extent of cortical infarction and ongoing neuronal loss after hypoxia-ischemia in rats.

Treatment with insulin-like growth factor-1 has been demonstrated to reduce the extent of cortical infarction 5 days after hypoxic-ischemic brain injury. As neuronal death can be progressive and long lasting after initial injury, the present study examined the long-term effects of insulin-like growth factor-1 on late neuronal loss 20 days after hypoxic-ischemic injury, together with evaluating neurobehavioral outcome as assumed by somatosensory function. Unilateral brain injury was induced in adult rats by carotid artery ligation followed by 10 min of hypoxia (6% O2). A single dose of insulin-like growth factor-1 (50 microg) was administered intracerebroventricularly via a stereotaxically pre-fixed cannula 2 h after injury. A bilateral tactile stimulation test was used to examine the degree of somatosensory function at 3, 5, 10 and 20 days after the hypoxia in both insulin-like growth factor-1- (n=12) and its vehicle- (n=12) treated rats, along with sham-operated rats (n=9). Cortical infarction and percentage of selective neuronal loss in the cerebral cortex were examined 20 days after the hypoxic-ischemic injury in both treatment groups. Hypoxic-ischemic injury resulted in a significant delay in the time taken to contact the patch over the period examined (left/right ratio 5.1+/-0.79), particularly at 3 days (7.0+/-2.8) after the hypoxia, compared to sham-operated rats (1.1+/-0.9, P<0.05). The overall effect of insulin-like growth factor-1 in reducing the time taken to contact the patch was significant (P=0.03, 2.6+/-0.79) compared to the vehicle group. There was a trend towards a reduction of cortical infarction after insulin-like growth factor-1 treatment (P=0.058), however insulin-like growth factor-1 significantly reduced the percentage of selective neuronal loss (P=0.027) 20 days following the hypoxia. From these data we suggest that insulin-like growth factor-1 improves somatosensory function by reducing both the extent of cortical infarction and ongoing progressive neuronal death during brain recovery from hypoxic-ischemic injury.

Animals↗

PubMed2001

Cvt19 is a receptor for the cytoplasm-to-vacuole targeting pathway.

Cvt19 is specifically required for the transport of resident vacuolar hydrolases that utilize the cytoplasm-to-vacuole targeting (Cvt) pathway. Autophagy (Apg) and pexophagy, processes that use the majority of the same protein components as the Cvt pathway, do not require Cvt19. Cvt19GFP is localized to punctate structures on or near the vacuole surface. Cvt19 is a peripheral membrane protein that binds to the precursor form of the Cvt cargo protein aminopeptidase I (prAPI) and travels to the vacuole with prAPI. These results suggest that Cvt19 is a receptor protein for prAPI that allows for the selective transport of this protein by both the Cvt and Apg pathways.

Aminopeptidases↗

PubMed2001

Surface modification of polyurethane for promotion of cell adhesion and growth 1: surface photo-grafting with N,N-dimethylaminoethyl methacrylate and cytocompatibility of the modified surface.

Functional polyurethane (PU) surface was prepared by photo-grafting N,N-dimethylaminoethyl methacrylate (DMAEM) onto the membrane surface. Grafting copolymerization was conducted by the combined use of the photo-oxidation and irradiation grafting. PU membrane was photo-oxidized to introduce the hydroperoxide groups onto the surface, then the membrane previously immersed in monomer solution was irradiated by UV light. The X-ray photoelectron spectroscopy and water contact angle characterized the grafted copolymers and verified the occurrence of graft copolymerization. The results showed that UV irradiation could realize the graft copolymerization effectively. The grafted membrane showed minimal surface morphology. Human umbilical vein endothelium (HUVE) cells were seeded on the grafted surfaces. The performance of the surface in cell attachment correlated with the content of oxygen and nitrogen. Cells were spread more extensive and grown faster on the surface with lower degree of grafting.

Journal Article↗

PubMed2001

Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris.

Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

Amino Acid Sequence↗

PubMed2001

Insulin-like growth factor-1 reduces postischemic white matter injury in fetal sheep.

Insulin-like growth factor-1 (IGF-1) is known to be important for oligodendrocyte survival and myelination. In the current study, the authors examined the hypothesis that exogenous IGF-1 could reduce postischemic white matter injury. Bilateral brain injury was induced in near-term fetal sheep by 30 minutes of reversible carotid artery occlusion. Ninety minutes after ischemia, either vehicle (n = 8) or a single dose of 3 microg IGF-1 (n = 9) was infused intracerebroventricularly over 1 hour. White matter changes were assessed after 4 days recovery in the parasagittal intragyral white matter and underlying corona radiata. Proteolipid protein (PLP) mRNA staining was used to identify bioactive oligodendrocytes. Glial fibrillary acidic protein (GFAP) and isolectin B-4 immunoreactivity were used to label astrocytes and microglia, respectively. Myelin basic protein (MBP) density and the area of the intragyral white matter tracts were determined by image analysis. Insulin-like growth factor-1 treatment was associated with significantly reduced loss of oligodendrocytes in the intragyral white matter (P < 0.05), with improved MBP density (P < 0.05), reduced tissue swelling, and increased numbers of GFAP and isolectin B-4 positive cells compared with vehicle treatment. After ischemia there was a close association of PLP mRNA labeled cells with reactive astrocytes and macrophages/microglia. In conclusion, IGF-1 can prevent delayed, postischemic oligodendrocyte cell loss and associated demyelination.

Animals↗

PubMed2001

Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase.

The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth inhibition caused by androgen ablation. (ii) Tyrosine kinases are involved in neurotrophic factor-mediated AR activation and, as such, may serve as targets of future therapeutics, to be used in conjunction with current antihormone and antineuropeptide therapies.

Androgens↗

PubMed2001

The premature fetus: not as defenseless as we thought, but still paradoxically vulnerable?

Traditionally, it has been believed that the cardiovascular and hormonal responses to asphyxia in preterm fetuses are immature, and this immaturity contributes to their apparent vulnerability to neural injury. However, these data were derived from studies using relatively mild insults, which did not allow for the greater cardiac glycogen reserves and anaerobic capacity of the brain near midgestation. Here, we review the maturation of the cardiovascular and cerebrovascular and cerebral responses to asphyxia in experimental animals and how these relate to the apparent vulnerability of the human premature brain. Most such investigations have been performed in the chronically instrumental fetal sheep. Recent studies have demonstrated that the premature fetus has highly adaptive and relatively mature responses to asphyxia, and that in absolute terms the preterm brain is very resistant to asphyxial injury. These data suggest that the premature fetus is able to survive much more prolonged periods of asphyxia than the near-term fetus, but that, paradoxically, such survival is associated with exposure to prolonged periods of hypotension and hypoperfusion and consequently greater risk of severe neural damage.

Animals↗

PubMed2001

Size and structure effects on centrifugal dewatering of digested sewage sludge.

The application of light scattering over small angles for the determination of digested sludge floc size and structure and its relationship with dewaterability is investigated. It appears that improved dewatering corresponds with lower floc fractal dimension (a more open structure) and a smaller proportion of fine particles. The initial increase in fractal dimension with increasing polymer dose for the digested sludge is most likely due to more efficient aggregation of the finer particles and the resulting formation of denser particle aggregates. A large colloidal fractal of the digested sludge (< 10 microm) appears to be less negative than the bulk digested sludge. This suggests that the fine particles will react differently and possibly less aggressively to the cationic polymer than the larger and more negative particles. The higher negative charge associated with the larger particles might be related to greater levels of highly negatively charged extracellular polysaccharides (EPS) adsorbed to the flocs or could result from the association of FeS with the finer fraction. The appearance of much greater levels of fine particles after digestion suggests that the flocs have undergone disintegration. Whether this is due to reduced biological efficiency within the digestor or iron reduction under the anaerobic conditions is not known for certain, although no indication of prolonged stress in the digesters could be found from plant performance data.

Centrifugation↗

PubMed2001

[Study on the correlation of estrogen receptor gene polymorphism to bone mineral density of radius in Chinese Han postmenopausal women in Beijing].

OBJECTIVE: To investigate the distribution of estrogen receptor (ER) gene polymorphism in postmenopausal Han women in Beijing as well as its correlation with bone mineral density (BMD) of radius. METHODS: Xba I, and Pvu II polymorphism of ER gene were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 99 healthy postmenopausal Han women. BMD of distal (spongy bone) and proximal (compact bone) radius were determined by dual-energy X-ray absorptiometry (DEXA) as well. The relationship between BMD and polymorphism of ER gene was analyzed by variance analysis. RESULTS: Pvu II polymorphism of ER gene is not associated with BMD of both sites of radius (P > 0.05); while Xba I polymorphism of ER gene is significantly associated with BMD spongy and compact bone of radius (P < 0.05). Women with XX genotype have the lowest BMD T-score of -1.55 +/- 0.37 for the compact and -2.54 +/- 0.38 for the spongy, and those with xx genotype have the highest BMD T-score of -0.95 +/- 0.24 for compact and -1.74 +/- 0.16 for spongy. CONCLUSION: There is a significant correlation of Xba I polymorphism of ER gene with BMD of distal and proximal radius. It is indicated that the difference in ER genotypes among individuals might influence the development of osteoporosis.

Asian People↗

PubMed2001

Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes.

OBJECTIVES: To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells. METHODS: Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the "ping-pong" technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells. RESULTS: The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6 x 10(5) cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7 microgram/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4 ng.ml-1 x 10(6) cells-1 x 2 days-1. CONCLUSION: The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.

3T3 Cells↗