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X Liu

Publications and source records attributed to X Liu.

At least 1,153 records · Page 64Linked to original sources
PubMed1997

Fludarabine triphosphate inhibits nucleotide excision repair of cisplatin-induced DNA adducts in vitro.

Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.

Adenosine Monophosphate↗
PubMed1997

Alpha isoform of calcium-calmodulin dependent protein kinase II (CAM II kinase-alpha) restricted to excitatory synapses in the CA1 region of rat hippocampus.

Pre-embedding immunoperoxidase staining for CAM II kinase-alpha and post-embedding immunogold staining for glutamate and GABA, were used to reveal the subcellular distribution of CAM II kinase-alpha at transmitter-characterized synapses in the CA1 region of rat hippocampus. Immunoelectron microscopy showed that the majority of CAM II kinase-alpha-immunostained neuronal profiles were dendritic spines presumably derived from pyramidal cells. CAM II kinase-alpha immunoreactivity was mainly localized in postsynatic densities associated with glutamatergic axon terminals. No CAM II kinase-alpha immunoreactivity was detected in GABA-immunoreactive profiles or at GABAergic synapses. This study provides morphological evidence that CAM II kinase-alpha is involved only in excitatory neuronal transmission in the CA1 region. The enzyme is unlikely to be involved in plasticity at GABA synapses.

Animals↗
PubMed1997

Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis.

Therapeutic efficacy of interleukin-10 administration in colonic inflammation was assessed in rats. Following intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS), subcutaneous administration of 1-1000 micrograms/kg per day interleukin-10, or a placebo (0.9% NaCl) was commenced and continued for 5 days. Interleukin-10 administered at 1, 10 and 100 micrograms/kg per day significantly reduced myeloperoxidase activity by 34, 57, and 28%, respectively, compared to the placebo-treated group, which was paralleled by an attenuation of colonic tumor necrosis factor alpha (TNF-alpha) content. In contrast, the severity of mucosal necrosis was not affected by interleukin-10 administration at the dose range used. In addition, the 10-fold elevation in nitric oxide release, 5-fold rise in colonic nitrite production and enhanced expression of inducible nitric oxide synthase, associated with TNBS colitis, was not suppressed by interleukin-10. Interleukin-10 gene expression was elevated during the first 14 days of TNBS colitis. We conclude that 5 days administration of interleukin-10 in TNBS colitis displays mild anti-inflammatory properties which were not mediated via a nitric oxide-dependent pathway, but may involve TNF-alpha.

Animals↗
PubMed1997

Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution.

Programmed cell death (PCD) of mammary alveolar cells during involution commences within hours of the end of suckling. Locally, milk accumulates within alveolar lumens; systemically, levels of lactogenic hormones fall. Four experimental models were used to define the role of local factors as compared with systemic hormones during the first and second stages of involution. In three models, milk release was disrupted in the presence of systemic lactogenic hormones: (i) sealing of the teats, (ii) mammary gland transplants that cannot release milk due to the absence of a teat connection, and (iii) inactivation of the oxytocin gene. The ability of systemic hormones to preserve lobular-alveolar structure without blocking PCD was illustrated using a fourth transgenic model of lactation failure. During the first stage of involution, local signals were sufficient to induce alveolar PCD even in the presence of systemic lactogenic hormones. PCD coincided with bax induction, decreased expression of milk proteins, block of prolactin signal transduction through Stat5a and 5b, and activation of Stat3. The two stages of mammary gland involution are regulated by progressive gain of death signals and loss of survival factors. This study demonstrates that genetic events that occur during the first reversible stage are controlled by local factors. These mammary-derived death signals are dominant over protective effects related to systemic hormone stimulation.

Animals↗
PubMed1997

Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.

The aspartate-99 of secreted phospholipase A2 (PLA2) has been proposed to be critical for the catalytic mechanism and interfacial activation of PLA2. Aspartate-99 connects the catalytic machinery (including the catalytic diad, the putative catalytic waters W5 and W6, and the calcium cofactor) to the hydrogen-bonding network. The latter involves Y52, Y73, the structural water, and the N-terminal region putatively required for the interfacial activation. A triple mutant of bovine pancreatic PLA2 with substitutions aspartate plus adjacent tyrosine residues (Y52,73F/D99N) was constructed, its X-ray structure was determined, and kinetic characteristics were analyzed. The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994) Protein Sci. 3, 2082-2088]. Kinetic analysis demonstrated that these mutants possessed considerable catalytic activity with a k(cat) value of about 5% compared to WT. The values of the interfacial Michaelis constant were also little perturbed (ca. 4-fold lower for D99N and marginally higher for Y52,73F/D99N). The results taken together suggest that the hydrogen-bonding network is not critically important for interfacial activation. Instead, it is the chemical step that is perturbed, though only modestly, in the mutants.

Amino Acid Sequence↗
PubMed1997

Epstein-Barr virus permissively infects human syncytiotrophoblasts in vitro and induces replication of human T cell leukemia-lymphoma virus type I in dually infected cells.

Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1), as well as human T-cell leukemia-lymphoma virus type I (HTLV-I), may interact in the pathogenesis of human retroviral infections. The placental syncytiotrophoblast layer represents a barrier protecting the fetal compartment from exposure to retroviruses. We studied the interactions of EBV with HIV-1 and HTLV-I in human term syncytiotrophoblast cells to investigate the significance of double infections in transplacental transmission of human retroviruses. We found that syncytiotrophoblast cells could be productively infected with EBV. Dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. In contrast, the restricted permissiveness of syncytiotrophoblasts for HIV-1 was not influenced by coinfection of the cells with EBV. Infection of syncytiotrophoblast cells with EBV, but not HTLV-I, induced interleukin-2 and interleukin-6 secretion, and augmented secretion occurred on coinfection with both viruses. Coinfection of syncytiotrophoblast cells with EBV and HTLV-I induced tumor necrosis factor-beta and transforming growth factor-beta 1 secretion, but infection with either virus alone did not lead to secretion of these cytokines. Permissive replication cycle of HTLV-I was induced by the EBV immediate-early gene product Zta. Pseudotype formation between EBV and HTLV-I in coinfected syncytiotrophoblast cells was not found. Our data suggest that activation of HTLV-I gene expression by EBV in coinfected syncytiotrophoblast cells may be a mechanism for transplacental transmission of HTLV-I.

Animals↗
PubMed1997

Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development.

Type III collagen is a fibrillar forming collagen comprising three alpha1(III) chains and is expressed in early embryos and throughout embryogenesis. In the adult, type III collagen is a major component of the extracellular matrix in a variety of internal organs and skin. Mutations in the COL3A1 gene have been implicated as a cause of type IV Ehlers-Danlos syndrome, a disease leading to aortic rupture in early adult life. To directly study the role of Col3a1 in development and disease, we have inactivated the Col3a1 gene in embryonic stem cells by homologous recombination. The mutated allele was transmitted through the mouse germ line and homozygous mutant animals were derived from heterozygous intercrosses. About 10% of the homozygous mutant animals survived to adulthood but have a much shorter life span compared with wild-type mice. The major cause of death of mutant mice was rupture of the major blood vessels, similar to patients with type IV Ehlers-Danlos syndrome. Ultrastructural analysis of tissues from mutant mice revealed that type III collagen is essential for normal collagen I fibrillogenesis in the cardiovascular system and other organs.

Animals↗
PubMed1997

Insulin-like growth factor-I treatment reduces immune cell responses in acute non-demyelinative experimental autoimmune encephalomyelitis.

To test the effects of insulin-like growth factor-I (IGF-I) on clinical deficits, lesion severity, and immune cell response in acute, non-demyelinative experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats by passive transfer of an MBP-reactive T lymphocyte line. Four days after receiving 5 x 10(5) MBPL-1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 micrograms IGF-I i.v. twice daily for 6 days, and the other 10 received the same volume of 0.89% NaCl. Pairs of rats were sacrificed after 4 days and 6 days of IGF-I and placebo treatment and spinal cord sections were processed for immunostaining, in situ hybridization, and morphological examination. IGF-I treatment decreased clinical deficits, lesion numbers, and lesion areas significantly. Numbers of CD4-positive T cells, alpha/beta TCR-positive cells, and ED-1-positive macrophages were also significantly reduced by IGF-I treatment. Similar reductions were found in our second trial, when 11 days of placebo and IGF-I injections began the day after transfer. No demyelination was observed in either toluidine blue-stained semithin sections or sections immunostained with an antibody raised against myelin basic protein (MBP). We conclude that IGF-I-induced reductions in immune cell responses can occur in the absence of demyelination and are of major importance in decreasing clinical deficits and lesion severity in EAE. If IGF-I has similar effects in multiple sclerosis, we think that it will be useful therapeutically.

Animals↗
PubMed1997

Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles.

The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-term biological assays or required highly purified test articles. These methods were inadequate for optimizing adenovirus production and purification. This assay can quantitate viral particles in either crude lysates or highly pure samples. It can be used to assess particles in both dilute and concentrated samples over a wide dynamic range. Moreover, the population of viral particles eluted in the peak contains most of the infectious virions. This assay is a sensitive technique that overcomes the limitations of previous methods. It provides an essential tool to accomplish process optimization.

Adenoviridae↗
PubMed1997

Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.

Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.

Apoproteins↗
PubMed1997

Protection of nitrergic neurotransmission by and colocalization of neural nitric oxide synthase with copper zinc superoxide dismutase.

This study examined in the rat anococcygeus muscle the tissue distribution of copper zinc superoxide dismutase, the activity of CuZn SOD, and the role of CuZn SOD in protecting nitric oxide from destruction by superoxide anion. Immunohistochemical studies revealed intense staining for CuZn SOD in neuronal nitric oxide synthase-containing nerves. Muscle strips contained 1081 +/- 300 units SOD g-1 wet tissue (mean +/- S.E.M., n = 5). Diethyldithiocarbamate (2 mM) inhibited CuZn SOD activity in supernatant fractions of muscle homogenates by 34% (P < 0.01, n = 5), an effect reversed by CuCl2 (2 mM). In control conditions, electrical field stimulation of nitrergic inhibitory nerves evoked a 61.5 +/- 10.5% (n = 10) relaxation against guanethidine (30 microM)-induced tone. Relaxation evoked by nitrergic inhibitory nerves was neither potentiated by exogenous CuZn SOD (10-1000 U ml-1) nor reduced by the O-2-generator, pyrogallol (30 microM). When diethyldithiocarbamate (2 mM) was present, stimulation of nitrergic inhibitory nerves evoked a 51.7 +/- 10.8% (P < 0.05, n = 10) relaxation against guanethidine (30 microM)-induced tone. Addition of pyrogallol (30 microM) to diethyldithiocarbamate-treated (2 mM for 30 min) muscle strips further reduced nerve-evoked relaxation to 30.7 +/- 7.6% (P < 0.01, n = 10). The inhibitory effect of pyrogallol was reversed by exogenous CuZn SOD (100 U ml-1). Diethyldithiocarbamate (2 mM) had no effect on relaxation evoked by exogenous NO (1 microM). The data indicate that CuZn SOD is present in rat anococcygeus muscle, that it is colocalized with nNOS in the nitrergic nerves, and that it protects NO from destruction by O-2.

Animals↗
PubMed1997

The analgesic domain of IL-2.

Human recombinant interleukin-2 (IL-2) has been showed to exert an analgesic effect and some experiments suggested that opioid receptors were involved. Because the aromatic residues at the N-termini of opioid peptides are crucial for their binding to the opioid receptors, all nine aromatic residues in IL-2 were mutated by site-directed mutagenesis and both the analgesic activity and the immune activity of the wild-type and mutated IL-2 were tested. The result indicated that there exists a putative analgesic function domain in IL-2, in which Phe44, Tyr45, Tyrl07, and Phell7 were essential. These four residues are located close together at the tertiary structure level of IL-2.

Analgesics↗
PubMed1997

Stat5a is mandatory for adult mammary gland development and lactogenesis.

Prolactin (PRL) induces mammary gland development (defined as mammopoiesis) and lactogenesis. Binding of PRL to its receptor leads to the phosphorylation and activation of STAT (signal transducers and activators of transcription) proteins, which in turn promote the expression of specific genes. The activity pattern of two STAT proteins, Stat5a and Stat5b, in mammary tissue during pregnancy suggests an active role for these transcription factors in epithelial cell differentiation and milk protein gene expression. To investigate the function of Stat5a in mammopoiesis and lactogenesis we disrupted this gene in mice by gene targeting. Stat5a-deficient mice developed normally and were indistinguishable from hemizygous and wild-type littermates in size, weight, and fertility. However, mammary lobuloalveolar outgrowth during pregnancy was curtailed, and females failed to lactate after parturition because of a failure of terminal differentiation. Although Stat5b has a 96% similarity with Stat5a and a superimposable expression pattern during mammary gland development it failed to counterbalance for the absence of Stat5a. These results document that Stat5a is the principal and an obligate mediator of mammopoietic and lactogenic signaling.

Animals↗
PubMed1997

Identification of missense mutations in the Norrie disease gene associated with advanced retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity (ROP) is a retinal vascular disease occurring in infants with short gestational age and low birth weight and can lead to retinal detachment (ROP stages 4 and 5). X-linked familial exudative vitreoretinopathy is phenotypically similar to ROP and has been associated with mutations in the Norrie disease (ND) gene in some cases. OBJECTIVE: To determine if similar mutations in the ND gene may play a role in the development of advanced ROP. METHODS: Clinical examination and molecular genetic analysis were performed on 16 children, including 2 dizygotic and 1 monozygotic twin pairs, and their parents from 13 families. RESULTS: Sequencing of the amplified products revealed missense mutations (R121W and L108P) in the third exon of the ND gene in 4 patients. These mutations were not present in an unaffected premature twin, 2 children with regressed stage 3 ROP, the parents, or in 50 unrelated healthy control subjects. CONCLUSION: These findings suggest that mutations in the ND gene may play a role in the development of severe ROP in premature infants.

DNA Mutational Analysis↗
PubMed1997

High interferon alpha levels in placenta, maternal, and cord blood suggest a protective effect against intrauterine herpes simplex virus infection.

Interferons (IFN) are produced by the placenta during pregnancy, and they can be detected in the maternal and fetal blood. Although the antiviral potential of IFNs is well established, it remains unclear whether the IFNs associated with pregnancy can prevent transplacental spread of viral infection. The present study was undertaken in order to determine the possible protective effect of placentally produced IFN-alpha on fetal acquisition of herpes simplex virus (HSV). Nine mothers with a known history of genital HSV infection were studied. In five cases IFN-alpha was detected in the placenta, maternal, and fetal blood, whereas in three cases IFN-alpha could not be detected. in the remaining case, IFN-alpha was found only in the maternal blood. As corroborated by the serological evidence of early HSV infection in the cord blood, the single case of vertical HSV transmission was observed in the group of IFN nonproducers. Furthermore, virus transmission did not occur in cases where IFN-alpha was present in the placenta and simultaneously in the maternal and fetal circulations. Thus, the present data indicate that high levels of IFN during pregnancy may protect the fetus from acquiring a possibly fatal intrauterine HSV infection.

C-Reactive Protein↗
PubMed1997

Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells.

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome.

Blotting, Western↗
PubMed1997

Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission.

The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.

Animals↗