Biomedical subjects

L Qin

Publications and source records attributed to L Qin.

At least 19 recordsLinked to original sources

PubMed2001

Differential IL-10R1 expression plays a critical role in IL-10-mediated immune regulation.

In this study, we characterized the differential receptor-binding specificity, affinity, and Janus kinase-STAT activation of cellular IL-10 (cIL-10) compared with viral IL-10 (vIL-10). Only cells expressing IL-10R1 bind human IL-10 or vIL-10. IL-10R2 does not bind to cIL-10 or vIL-10 alone and its presence does not enhance the receptor-binding affinity of cIL-10 or vIL-10, but it is essential for both cIL-10- and vIL-10-mediated signal transduction and immune regulation. Responses initiated by cIL-10 and vIL-10 were compared in B cell and mast cell lines, and demonstrated that the inability of vIL-10 to stimulate immune responses, as compared with human IL-10, is due to failure to initiate signaling. Absent signal transduction is due to low level expression of cell surface IL-10R1, since overexpressing IL-10R1 allows vIL-10 to initiate cIL-10-like signals and subsequent biological responses. These results are similar in primary cells, since splenocytes respond to both cIL-10 and vIL-10, while thymocytes respond only to cIL-10 and have very low mouse IL-10R1 but not mouse IL-10R2 expression. These data demonstrate that IL-10R1 expression plays a critical role in determining whether cells respond to IL-10. Modulation of cell surface IL-10R1 density might be an important mechanism for determining whether IL-10 leads to immunostimulation or immunosuppression in vivo.

Animals↗

PubMed2001

Hippocampal mossy fibers induce assembly and clustering of PSD95-containing postsynaptic densities independent of glutamate receptor activation.

Factors that regulate the formation, spatial patterning, and maturation of CNS synapses are poorly understood. We used organotypic hippocampal slice cultures derived from developing (P5-P7) rat to test whether synaptic activity regulates the development and organization of postsynaptic structures at mossy fiber (MF) giant synapses. Antibodies to a prominent postsynaptic density (PSD) scaffold protein, PSD95, identified large (>1 microm) and irregularly shaped PSD assemblies that codistributed with synapsin-I or metabotropic glutamate receptor 7b (mGluR7b) -immunolabeled MF terminals in area CA3. To investigate the spatial organization of synaptic PSDs on individual pyramidal cells, neurons in slice cultures were transfected with a vector encoding a GFP-PSD95 fusion protein. Confocal three-dimensional reconstructions revealed clusters of PSDs along proximal dendrites of transfected pyramidal neurons in area CA3, but not in CA1. Clusters averaged 7.6 microm in length (range, 2.2-29 microm) and contained up to 35 individual PSDs (mean, 8.3). PSD clusters failed to form when slices were cultured without MFs, indicating that MFs induce cluster assembly. Chronic blockade of N-methyl-D-apartate- and AMPA/kainate-type glutamate receptors did not disrupt MF targeting or de novo formation of PSD clusters with a normal distribution on target cells. Additionally, glutamate receptor blockers did not alter the ultrastructural development of MF giant synapses containing multiple puncta adherens-like junctions and asymmetric synaptic junctions at dendritic shaft and spine domains, respectively. The results indicate that MF axons can induce the assembly and clustering of PSD95-containing postsynaptic complexes, displaying a normal subcellular and tissue distribution, by mechanisms that are independent of ionotropic glutamate receptor activation.

Animals↗

PubMed2001

Role of CtBP in transcriptional repression by the Drosophila giant protein.

The giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including knirps, Krüppel, and snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. We studied giant repression activity in a CtBP mutant and find that this cofactor is required for giant repression of some, but not all, genes. While targets of giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Krüppel gene, in which one enhancer is repressed by giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the giant repression activity, we have identified a minimal repression domain within giant that encompasses residues 89-205, including an evolutionarily conserved region bearing a putative CtBP binding motif.

Alcohol Oxidoreductases↗

PubMed2001

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Animals↗

PubMed2001

Parvalbumin expression is downregulated in rat fast-twitch skeletal muscles during aging.

In this study, the protein expression profile of extensor digitorum longous (EDL) and Soleus (SOL) muscles, representing fast- and slow-twitch skeletal muscles, respectively, was established using high resolution two-dimensional electrophoresis (2-DE). One protein spot was found uniquely expressed in EDL muscle. N-terminal sequence analysis identified the protein as parvalbumin. Parvalbumin is a high affinity calcium binding protein that regulates muscle contraction and relaxation. Our experiments revealed that parvalbumin expression in EDL muscle was down-regulated during aging. In addition, high-intensity exercise could reverse this age-related change. Soleus muscles do not normally express parvalbumin, but high-intensity exercise could ectopically induce its expression in both young and old SOL muscles. We have also confirmed our 2-DE findings by immunohistochemistry on muscle sections. Our results suggest that high-intensity training could be used to improve muscle functions during aging because parvalbumin play an important role in regulating skeletal muscle contraction and relaxation.

Aging↗

PubMed2001

Viral IL-10-induced immunosuppression requires Th2 cytokines and impairs APC function within the allograft.

Previous reports demonstrated that retroviral mediated gene transfer of viral IL-10 (vIL-10) prolongs allograft survival by decreasing donor-specific cytotoxic T lymphocyte precursor (CTLp) and IL-2-secreting helper T lymphocyte precursor (HTLp) frequency within graft-infiltrating cells (GIC). This report now shows that vIL-10 efficacy is dependent on CD4(+) T cells, suggesting that immunosuppression may involve a switch from a Th1 to a Th2 alloresponse. In support of this, anti-IL-4 or anti-murine IL-10 (anti-mIL-10) mAbs, but not anti-IFN-gamma mAb, administered at the time of vIL-10 gene transfer prevents enhanced graft survival. Because Th switching involves APC function, GIC were examined for their ability to present alloantigen. GIC from vIL-10-treated grafts were shown to be mostly of recipient (CBA) origin, yet were unable to elicit alloproliferative responses from donor type (C57BL/6) or third party (BALB/c) responders. The inability of vIL-10-treated GIC to stimulate the MLR was not due to the generation of negative regulatory cells or the production of immunosuppressive cytokines such as IL-4, mIL-10, or TGFbeta. Using fractionated GIC subpopulations, the number of recipient type cells in the allografts was modestly reduced by vIL-10 gene transfer, while maintaining both APC phenotype and alloantigen presenting function. Conversely, after vIL-10 gene transfer, donor type GIC were unable to participate in direct alloantigen presentation. Therefore, local immunosuppression induced by vIL-10 gene transfer is CD4 T cell and IL-4 and mIL-10 dependent, and impairs direct alloantigen presentation through an alteration of donor type APC function.

Animals↗

PubMed2001

Osteopenia in adolescent idiopathic scoliosis: a histomorphometric study.

STUDY DESIGN: Bone biopsies from iliac crest and spinous process of adolescent idiopathic scoliosis patients were obtained intraoperatively for histology and histomorphometric analysis. OBJECTIVES: To study the histologic features of cancellous bone and to correlate the histomorphometric variables with preoperative bone mineral density in patients with adolescent idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: Low bone mineral density has been reported in adolescent idiopathic scoliosis. However, there is limited information about the histopathologic changes. METHODS: Undecalcified and decalcified bone specimens from iliac crest and spinous process of adolescent idiopathic scoliosis patients obtained intraoperatively were stained with Goldner and hematoxylin & eosin stain, respectively. Results were correlated with bone mineral density of the lumbar spine (L2-L4) and proximal femur measured before surgery. RESULTS: Bone histology showed significant less osteocyte count in the trabecular bone characterized with smooth and continuous borders in patients with adolescent idiopathic scoliosis. Histomorphometry confirmed the lower static parameters. The results correlated well with the decreased bone mineral density. CONCLUSION: Bone biopsy study suggested disturbance of bone turnover in patients with adolescent idiopathic scoliosis. The abnormal metabolism might contribute to the low bone mineral density and play an important role in the etiology and pathogenesis of adolescent idiopathic scoliosis.

Adolescent↗

PubMed2001

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli.

Escherichia coli modulates its porin expression through a histidine kinase, EnvZ, and its cognate response regulator, OmpR. EnvZ is a bifunctional enzyme that possesses both OmpR kinase and phosphorylated OmpR (OmpR-P) phosphatase activities and thus controls the cellular level of OmpR-P. In an in vitro-assay system, the addition of OmpR to the reaction mixture consisting of the cytoplasmic domain of EnvZ (EnvZc) and ATP produces a barely detectable amount of OmpR-P because of the dual activities of EnvZ. Here we report that DNA fragments containing the upstream promoter regions of the porin genes (ompF and ompC) can shift the equilibrium between OmpR and OmpR-P dramatically toward OmpR-P. Among the four reactions occurring in the mixture, only the EnvZ phosphatase activity was inhibited severely by the specific DNA, in contrast to the previous report by Kenney and her associates that DNA stimulates OmpR phosphorylation by EnvZ [Ames, S. K., Frankema, N. & Kenney, L. J. (1999) Proc. Natl. Acad. Sci. USA 96, 11792-11797]. The autophosphorylation of EnvZc and the phosphotransfer from phosphorylated EnvZc to OmpR were not affected by DNA, whereas the autodephosphorylation of OmpR-P was inhibited slightly. We propose that the apparent inhibitory effect of DNA on the EnvZ phosphatase function is caused by sequestrating OmpR-P from the reaction as a result of OmpR-P binding to DNA.

Bacterial Outer Membrane Proteins↗

PubMed2001

Identification of TNF-alpha-sensitive sites in HCMVie1 promoter.

Viral vectors using the human cytomegalovirus immediate-early promoter (HCMVie1 promoter) are potentially efficient tools for gene delivery in vivo to diverse cell types. We previously demonstrated that two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma), inhibited transgene expression from this promoter in skeletal and cardiac myocytes. In this study, electrophoretic mobility shift assays (EMSAs) were performed to identify the TNF-alpha response elements from the HCMVie1 promoter. The results show that TNF-alpha enhances the interaction of nuclear proteins from the C2C12 myocyte line with a single restricted segment of the HCMVie1 promoter. In vitro DNase I footprinting defined precisely the sites of interaction to two elements: nucleotides -1 to 0 and +24 to +36 relative to a transcription initiation cap homologous in the HCMVie1 promoter. These sites contain homologous sequences for cap initiation site (82%) and NFkappaB (62%) sites, respectively. Specificity was further ascertained by competitive EMSAs with wild-type and mutant oligonucleotide probes. Southwestern blotting showed that three proteins (45, 30, and 20 kDa) bound to this TNF-alpha-sensitive element, separately. However, EMSAs failed to prove a role for Yin Yang-1 (YY-1), NFkappaB (p65), or NFkappaB (p50) in binding to these sites. Our results provide evidence for two novel sites in the HCMVie1 promoter that are targets for TNF-alpha enhanced binding of transcription factors.

Antigens, Viral↗

PubMed2001

TNFalpha and IFNgamma induced by innate anti-adenoviral immune responses inhibit adenovirus-mediated transgene expression.

The transient nature of adenovirus-mediated transgene expression has been attributed to adaptive immune responses to adenoviral proteins and transgene products. However, the cytokines interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) inhibit transgene expression from adenoviral vectors in vitro by a transcription-related mechanism, and their early induction following vector administration in vivo suggests a contribution of innate immunity in regulating transgene expression. In this study, the significance of cytokine expression and its relation to adaptive and innate immunities were determined in TNFalpha-knockout mice, IFNgamma-knockout mice, or anti-IFNgamma mAb-injected animals. Adenoviral LacZ reporter expression directed by human cytomegalovirus (HCMV) promoters was greater in magnitude and duration than that by the murine CMV (MCMV) promoter. beta-Galactosidase reporter gene expression up to day 7 was greater in cytokine-deficient animals compared with wild type. Decrements in transgene expression occurred in advance of adaptive immune responses and were not due to alterations in specific adaptive immunity or vector clearance in cytokine-depleted mice. We conclude that TNFalpha and IFNgamma inhibit early adenovirus-mediated transgene expression by HCMV and MCMV promoters in vivo. Cytokine inhibition of expression is independent of adaptive immunity and is likely secondary to innate immune responses to adenovirus infection.

Adenoviridae↗

PubMed2001

Measurement of the layered compressive properties of trypsin-treated articular cartilage: an ultrasound investigation.

An ultrasound-compression system has been developed for the study of the layered biomechanical properties of articular cartilage. Cartilage specimens harvested from the bovine patella groove, with and without trypsin digestion, were tested using this system. It was noted that a large ultrasound reflection can be detected in the interface of the trypsin digestion front. This ultrasound reflection signal was used to differentiate the deformations of different portions of the cartilage throughout its depth when a load was applied. The equilibrium compression moduli of the digested, undigested and entire portions of articular cartilage were measured. The modulus of the cartilage without any digestion was 660+/-230 kPa. After 1h digestion with 1 mg ml(-1) trypsin solution, the thickness of the digested portion was 0.50+/-0.06m, and the modulus of the entire cartilage layer changed to 125+/-42kPa. The moduli of the digested and undigested portions were 58+/-24 kPa and 470+/-31 kPa, respectively. Similar results were obtained for the cartilage with trypsin digestion for 2 h.

Animals↗

PubMed2001

Staining intensity of individual osteons correlated with elastic properties and degrees of mineralization.

Surface staining is widely used for histological studies involving undecalcified thick bone sections. Individual osteons, particularly newly formed ones stained with toluidine blue (TB), show various color intensities. We studied the correlations between TB color intensity and the differences in stiffness and degree of mineralization of individual osteons in undecalcified histological sections of goat tibial diaphysis, measured by scanning acoustic microscopy (SAM) and contact microradiography (CMR), respectively. Results showed that all three measurements correlated significantly with each other (r = 0.567 - 0.786; all P < or = 0.01). The TB surface staining intensity of individual osteons correlated better with the reflection coefficient (stiffness index) measured by SAM (r = 0.713) than with the aluminum step-wedge equivalent thickness measured on CMR micrographs (r = 0.567). The aluminum step-wedge equivalent thickness of individual osteons on CMR correlated slightly better with the SAM reflection coefficient (r = 0.786) than with the TB surface staining intensity (r = 0.713). The results of this study suggest that TB surface staining may be used as a simple method for indicating differences in stiffness and degree of mineralization in individual osteons in comparative histological studies.

Animals↗

PubMed2001

Antigenicity and predefined specificities of the multi-epitope vaccine in candidate consisting of neutralizing epitope and mutated epitopes suggested a new way against HIV-1 mutation.

A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.

AIDS Vaccines↗

PubMed2001

Meniscus and synovial membrane: an electronmicroscopic study on rabbits.

Seven healthy mature rabbits were used to study both the surface morphology of the meniscus using both transmission electronmicroscopy (TEM) and scanning electronmicroscopy (SEM) and articular cartilage of the femoral condyle using SEM. Results showed that the membrane covering the meniscus was structurally the extension of synovial membrane of the knee joint capsule. Additionally, the presence of canal-like openings over the membranes surface to the meniscus was noted, which were absent over the articular cartilage surface.

Animals↗

PubMed2001

Goats as an osteopenic animal model.

A large osteopenic animal model that resembles human osteoporotic changes is essential for osteoporosis research. This study aimed at establishing a large osteopenic animal model in goats. Twenty-five Chinese mountain goats were used in which they were either ovariectomized (OVX) and fed with a low-calcium diet (n = 16) or sham-operated (SHAM; n = 9). Monthly photodensitometric analysis on proximal tibial metaphysis and calcaneus was performed. Two iliac crest biopsy specimens obtained before and 6 months after OVX were used for bone mineral density (BMD) measurement with peripheral quantitative computed tomography (pQCT). Lumbar vertebrae (L2 and L7), humeral heads, and calcanei were collected for BMD measurement after euthanasia. The humeral heads and calcanei were used in biomechanical indentation test. BMD measurement showed a significant 25.0% (p = 0.006) decrease in BMD of the iliac crest biopsy specimens 6 months after OVX. It also was statistically significant when compared with the SHAM (p = 0.028). BMD at L2, L7, calcaneus, and humeral head reduced by 24-33% (p ranged from 0.001 to 0.011) when compared with the SHAM. Photodensitometry showed a continuous decrease in bone density after OVX. There were significant decreases of 18.9% in proximal tibial metaphysis (p = 0.003) and 21.8% in calcaneus (p = 0.023) in the OVX group 6 months postoperatively. Indentation test on the humeral head and calcaneus showed a significant decrease 52% (p = 0.006) and 54% (p = 0.001), respectively, in energy required for displacement of 3 mm in the OVX group compared with the SHAM group. The decreases correlated significantly to the decrease in BMD of the corresponding specimens (r2 = 0.439 and 0.581; p < 0.001 for both). In conclusion, this study showed that OVX plus a low-calcium diet could induce significant osteopenia and deterioration of mechanical properties of the cancellous bone in goats.

Animals↗

PubMed2001

Femtomolar concentrations of dynorphins protect rat mesencephalic dopaminergic neurons against inflammatory damage.

The hallmark of Parkinson's disease is the death of nigral dopaminergic neurons, and inflammation in the brain has been increasingly associated with the pathogenesis of this neurological disorder. Dynorphins are among the major opioid peptides in the striato-nigral pathway and are important in regulating dopaminergic neuronal activities. However, it is not clear whether dynorphins play a role in the survival of nigral dopaminergic neurons. We have recently demonstrated that lipopolysaccharide (LPS) activates the brain immune cells microglia, in vitro and in vivo, to release neurotoxic factors to degenerate dopaminergic neurons. The purpose of this study was to explore the neuroprotective effect of dynorphins in the inflammation-mediated degeneration of dopaminergic neurons in rat midbrain neuron-glia cultures. LPS-induced neurotoxicity was significantly reduced by treatment with ultra low concentrations (10(-13)--10(-15) M) of the kappa-opioid receptor agonist dynorphin A (1--17) or the receptor binding ineffective [des-Tyr(1)]dynorphin A (2--17), but not by U50488, a synthetic kappa-receptor agonist. The glia-mediated neuroprotective effect of dynorphins was further supported by the finding that femtomolar concentrations of dynorphins did not prevent the killing of dopaminergic neurons by 6-hydroxydopamine. However, ultra low concentrations of dynorphins inhibited LPS-induced production of superoxide. These results suggest a glia-mediated and conventional opioid receptor-unrelated mechanism of action for the neuroprotective effect of ultra low concentrations of dynorphins. Understanding the underlying mechanisms of action should further define the roles of dynorphins in the regulation of dopaminergic neurons and help devise novel strategies to combat neurodegenerative diseases.

Animals↗

PubMed2001

[Relationship between serum matrix metalloproteinase-2 and metastasis and recurrence following radical hepatic resection in hepatocellular carcinoma].

OBJECTIVE: To study the relationship between serum matrix metalloproteinase-2 (MMP-2) and metastasis and recurrence following radical hepatic resection in hepatocellular carcinoma (HCC). METHODS: Cox's proportional hazard model was used to determine the significant clinical factor of prognosis. Meanwhile, enzyme linked immunoadsordent assay (ELISA) and northern blot analysis were adopted to detect serum and tumor tissue levels of MMP-2 respectively in HCC patients. RESULTS: Cox's proportional hazard model showed that tumor size and tumor number were significant prognostic factors of HCC patients following hepatic resection (P=0.022 and P=0.040). According to this result, we divided HCC patients into high-tendency to metastasis recurrence and low-tendency to metastasis recurrence groups. Serum levels of MMP-2 in high-tendency to metastasis recurrence following radical hepatic resection group were significantly higher than those in low-tendency group (26.39ng/m+/-2.64ng/ml vs 24.86ng+/-1.95ng/ml, P<0.05). Serum levels of MMP-2 in HCC patients performed non-radical hepatic resection group were also significantly higher than those in HCC patients performed radical hepatic resection group (29.43ng+/-3.12 ng/ml vs 25.72ng+/-2.45ng/ml, P<0.01). Northern blot analysis revealed that the changes of MMP-2 level in the serum were consistent with those of its mRNA level in the tumor tissue of HCC patients. CONCLUSIONS: Serum MMP-2 level is an indicator for metastatic recurrence following radical hepatic resection in HCC. The high levels of MMP-2 in the serum are due to high MMP-2 mRNA expression in the tumor tissue of HCC patients.

Carcinoma, Hepatocellular↗

PubMed2000

Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types.

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.

Animals↗