Biomedical subjects

Hong Liu

Publications and source records attributed to Hong Liu.

At least 289 records · Page 16Linked to original sources

PubMed2003

Apoptotic action of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitro and in vivo.

BACKGROUND: Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Paradoxically, estrogen treatment inhibits the growth of long-term tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol. METHODS: Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor kappaB (NF-kappaB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided. RESULTS: Basal NF-kappaB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P =.004). When cultured with 1 microM raloxifene, MCF-7/Ral cells grew statistically significantly (P<.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G(2)/M phase of the cell cycle, decreased NF-kappaB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P<.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8- to 6.3-fold versus 0.5-fold, 95% CI = 0.3- to 0.8-fold for control treatment; P<.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P =.004 for raloxifene and P<.001 for tamoxifen). CONCLUSIONS: Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-kappaB activity, and increased Fas expression to induce apoptosis.

Animals↗

PubMed2003

Distinct molecular conformations of the estrogen receptor alpha complex exploited by environmental estrogens.

We have advanced the view that estrogens activate the estrogen receptor (ER) alpha complex differently. A group of planar (estradiol, genistein, and coumestrol) and nonplanar (methoxychlor and its mono- and didemethylated phenolic metabolites) environmental estrogens, which are all full estrogens in MCF-7 breast cancer cell proliferation assays, was shown to segregate discretely into planar and nonplanar groups. These groups were delineated using a novel assay of mutant ER cDNAs stably transfected into MDA-MB-231 cells and the activation of the transforming growth factor alpha target gene in situ that putatively describes the external shape of the ER complex. Planar compounds activate estrogen action through the two traditional activation functions (AFs), AF1 and AF2, in the ER. In contrast, nonplanar compounds can activate estrogen action through AF1 and the amino acids Asp-351 and Asp-538, which are exposed when helix 12 silences AF2. The observation that class I (planar) and class II (nonplanar) compounds have different mechanisms of estrogen action may have important implications for tissue selective modulation of the ER.

Cell Line, Tumor↗

PubMed2003

Generation of hair cells by stepwise differentiation of embryonic stem cells.

The increase in life expectancy is accompanied by the growing burden of chronic diseases. Hearing loss is perhaps the most prevalent of all chronic diseases. In addition to age-related hearing loss, a substantial number of cases of audiological impairment are either congenital in nature or acquired during childhood. The permanence of hearing loss is mainly due to the inability of the cochlear sensory epithelium to replace lost mechanoreceptor cells, or hair cells. Generation of hair cells from a renewable source of progenitors that can be transplanted into damaged inner ears is a principal requirement for potential cell replacement therapy in this organ. Here, we present an experimental protocol that enables us to routinely create inner ear progenitors from murine embryonic stem cells in vitro. These progenitors express a comprehensive set of marker genes that define the developing inner ear, in particular the organ's developing sensory patches. We further demonstrate that cells that express markers characteristic of hair cells differentiate from embryonic stem cell-derived progenitors. Finally, we show that these progenitors integrate into the developing inner ear at sites of epithelial injury and that integrated cells start expressing hair cell markers and display hair bundles when situated in cochlear or vestibular sensory epithelia in vivo.

Animals↗

PubMed2003

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner.

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.

Animals↗

PubMed2003

An alpha-1,3-mannosyltransferase of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus, distinguished by an elaborate polysaccharide capsule that is essential for its virulence. As part of an effort to understand the biosynthesis of this important structure, we initiated purification of an alpha-1,3-mannosyltransferase with appropriate specificity for a role in building the main capsule polysaccharide, glucuronoxylomannan. A pool of proteins that was 5,000-fold enriched in this activity included several polypeptides, which acted potentially as the catalytic protein. These were analyzed using sequence information and double-stranded RNA interference. Interference that targeted a sequence corresponding to part of a 46 kDa protein in the enriched fraction abolished the activity of interest and reduced the capsule on the affected cells. This gene was cloned and expressed in active form in Saccharomyces cerevisiae to confirm function, and was termed CMT1, for cryptococcal mannosyltransferase 1. CMT1 has no confirmed homologs in GenBank other than CAP59, a cryptococcal gene encoding a protein of unknown function that is required for capsule synthesis and virulence. The Cmt1p protein also co-purifies with a homolog of CAP64, a gene whose product has similarly been implicated in capsule synthesis and virulence. A strain disrupted in CMT1 was generated in C. neoformans; this had no effect on virulence in an animal model of cryptococcosis.

Amino Acid Sequence↗

PubMed2003

Maize-targeted mutagenesis: A knockout resource for maize.

We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

Base Sequence↗

PubMed2003

Pluripotent stem cells from the adult mouse inner ear.

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.

Animals↗

PubMed2003

[Exogenous rhCNTF inhibits myoblast differentiation of skeletal muscle of adult human in vitro].

To determine the role of recombinant human ciliary neurotrophic factor (rhCNTF) in myogenesis, we observed the effects of rhCNTF (0 10 ng/ml) on myoblast differentiation of adult human in vitro. The results showed that compared with the control group, the groups of 2.5 10 ng/ml rhCNTF treatment significantly inhibited myoblast differentiation (P<0.01), and the inhibition was dose-dependent and reversible. Western blots also indicated that compared with the control group, the expression of myogenin and p21, markers of myoblast differentiation phase, was significantly reduced (P<0.01), while Myf5 and desmin, markers of myoblast proliferative phase, significantly increased (P<0.01) in the groups of 2.5 10 ng/ml rhCNTF treatment. These findings demonstrate that exogenous rhCNTF can reversibly inhibit differentiation but permits proliferation of adult human myoblasts in vitro.

Adult↗

PubMed2003

Corpus callosum development in childhood-onset schizophrenia.

OBJECTIVE: Corpus callosum (CC) size and interhemispheric communication differences have been reported between patients with schizophrenia and normal controls. Childhood-onset schizophrenia (COS) is a severe form of the disorder that is continuous with later-onset disorder. Corpus callosal area was examined for COS at initial scan and prospectively through adolescence, and related to other developmental abnormalities for this group. METHOD: A total of 113 anatomic brain MRI scans were obtained from 55 COS (22 female) and 110 scans from 56 age- and gender-matched healthy volunteers (22 female), across ages 8-24. Baseline and prospective rescans were obtained at approximately 2-year intervals. The midsagittal areas for total corpus callosum and seven subregions were calculated using an automated system. Cross-sectional and longitudinal data were combined using mixed model regression analysis to compare developmental changes for the two groups. RESULTS: No diagnostic differences were seen at time of initial scan. Longitudinally, and in contrast to healthy volunteers, patients with schizophrenia showed a significant difference in developmental trajectory for the area of the splenium, both before (p=0.012) and after (p=0.05) adjustment for total cerebral volume. The area of the splenium becomes significantly smaller in COS, starting at about age 22. CONCLUSION: Patients with schizophrenia showed a significant difference in developmental trajectory for the splenial area, which seems to decline for COS. If replicated, this may reflect anticipated late occipital and extrastriate changes in brain regions.

Adolescent↗

PubMed2003

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis.

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

3T3 Cells↗

PubMed2003

Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency.

The growth of Lewis lung carcinoma (LLC) was sustained in plasminogen-deficient mice when transplanted into the dorsal skin but was dramatically suppressed in another anatomic location, the footpad. This unanticipated negative effect of plasminogen deficiency on footpad tumor growth was entirely relieved by superimposing a deficit in fibrinogen. This finding was not simply an unusual feature of LLC tumors--T241 fibrosarcoma growth in the footpad was also restricted by plasminogen deficiency in a fibrinogen-dependent manner. The probable mechanistic basis for suppression of tumor growth was revealed through transmission electron microscopy studies of tumor tissues. Occlusive microvascular thrombi were commonplace within footpad tumors from plasminogen-deficient mice, whereas no such lesions were observed within either dorsal skin tumors from plasminogen-deficient mice or footpad tumors from mice that also lacked fibrinogen. The data infer that tumor growth in the footpad of plasminogen-deficient mice is compromised as a function of the formation and persistence of vaso-occlusive thrombi that limit tumor blood supply. These studies indicate that plasminogen and fibrinogen can serve as critical determinants of tumor growth, but their relative importance is dependent on the tumor microenvironment. Furthermore, these studies suggest that one target of plasmin(ogen) relevant to tumor progression in vivo is intravascular fibrin.

Animals↗

PubMed2003

[Influence of modeling sodium on the dialysis disequilibrium syndrome in hemodialysis].

OBJECTIVE: To observe the influence of modeling sodium on the dialysis disequilibrium syndrome in hemodialysis. METHODS: Sixteen patients received hemodialysis for the first time. Eight of them received the hemodialysis in the order of common sodium, modeling sodium, common sodium, and modeling sodium, whereas the other 8 patients were treated in the order of modeling sodium, common sodium, modeling sodium, and common sodium. The symptoms of the dialysis disequilibrium syndrome were observed and blood sodium concentration was examined before and after the dialysis. RESULTS: The main symptoms of the dialysis disequilibrium syndrome were headache, nausea, vomiting, hypertension, and dyspnea. Modeling sodium hemodialysis could significantly decrease the occurrence of these symptoms (P < 0.05). The blood sodium concentration after the hemodialysis was not significantly changed compared with that before the hemodialysis (P > 0.05). CONCLUSION: Modeling sodium hemodialysis can effectively prevent the occurrence of the dialysis disequilibrium syndrome without increasing the natrium load of the patients.

Acid-Base Equilibrium↗

PubMed2003

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5.

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.

Animals↗

PubMed2003

ATIDB: Arabidopsis thaliana insertion database.

Insertional mutagenesis techniques, including transposon- and T-DNA-mediated mutagenesis, are key resources for systematic identification of gene function in the model plant species Arabidopsis thaliana. We have developed a database (http://atidb.cshl.org/) for archiving, searching and analyzing insertional mutagenesis lines. Flanking sequences from approximately 10 500 insertion lines (including transposon and T-DNA insertions) from several tagging programs in Arabidopsis were mapped to the genome sequence through our annotation system before being entered into the database. The database front end provides World Wide Web searching and analyzing interfaces for genome researchers and other biologists. Users can search the database to identify insertions in a particular gene or perform genome-wide analysis to study the distribution and preference of insertions. Tools integrated with the database include a graphical genome browser, a protein search function, a graphical representation of the insertion distribution and a Blast search function. The database is based on open source components and is available under an open source license.

Amino Acid Sequence↗

PubMed2003

Synthesis and structure-activity relationship of di-(3, 8-diazabicyclo[3.2.1]octane) diquaternary ammonium salts as unique analgesics.

Based on the structure characteristics of the lead compounds, 1, 1' octanedioyl-4, 4'-dimethyl-4, 4'-dibenzyl dipiperazinium dibromide (2) and 3, 8-disubstituted-3, 8-diazabicyclo [3.2.1]octanes (DBO), di-(3, 8-diazabicyclo [3.2.1]octane) diquaternary ammonium salts 3 a-c were designed and synthesized through a highly practical procedure. Target compounds 3 a-c and the hydrochloride salts of their precursors 10 a-c were evaluated for their in vivo analgesic and sedative activities. Interestingly, the introduction of an endoethylenic bridge in the piperazine of lead compound 2 causes loss of the analgesic activity and increases the toxicity dramatically. This result shows that the flexible conformation of piperazine in compound 2 is favorable for interaction with the receptor, and the quaternization of compounds 10 a-c is the main reason for the toxicity increase.

Analgesics↗

PubMed2003

Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina.

The neuroepithelial layer of the developing eyecup contains multipotential precursor cells that give rise to all of the neurons and the one glial cell type present in the adult retina. Patterning within the retinal neuroepithelium is regulated by cell intrinsic as well as cell extrinsic mechanisms. Although the identity of some of the signaling molecules that regulate retinal development is known, the function of many others, especially members of the Wnt family, has yet to be characterized in the context of retinal development. We undertook a comprehensive in situ hybridization analysis to examine the expression of Wnt pathway components in the developing and adult mouse neural retina. Our findings confirm and extend previous expression studies in mice and other vertebrates, as we show that Wnt-3, -5a, -5b, and -7b are expressed in the neural retina and that there is a dynamic pattern of Wnt receptor (Mouse frizzled [Mfz]) and Wnt antagonist (Secreted-frizzled-related protein [Sfrp]) gene expression in the embryonic and perinatal neural retina. Moreover, we show that Wnt-13 is expressed in the pigment epithelium overlying the distal part of the eyecup and the ciliary margin and that Mfz-4, -6, and -7 are expressed in different regions within the ciliary margin. To determine where activation of canonical Wnt signaling is occurring in the retina, we examined reporter gene expression in TCF/Lef-LacZ mice and we demonstrate that the highest levels of beta-gal activity are found in the ciliary margin, adjacent to and within the Wnt-13 expression domain, implicating Wnt-13 signaling in the development of the ciliary margin and its derivatives.

Animals↗

PubMed2003

Structure-based discovery of potassium channel blockers from natural products: virtual screening and electrophysiological assay testing.

Potassium ion (K(+)) channels are attractive targets for rational drug design. Based upon a three-dimensional model of the eukaryotic K(+) channels, the docking virtual screening approach was employed to search the China Natural Products Database. Compounds were ranked according to the relative binding energy, favorable shape complementarity, and potential of forming hydrogen bonds with the K(+) channel. Four candidate compounds found by virtual screening were investigated by using the whole-cell voltage-clamp recording in rat dissociated hippocampal neurons. When applied extracellularly, compound 1 markedly depressed the delayed rectifier K(+) current (I(K)) and fast transient K(+) current (I(A)), whereas compounds 2, 3, and 4 exerted a more potent and selective inhibitory effect on I(K). Intracellular application of the four compounds had no effect on both the K(+) currents.

Animals↗

PubMed2003

The low TN:TP ratio, a cause or a result of Microcystis blooms?

An enclosure experiment in the shallow, subtropical Lake Donghu, China, was performed in the summer of 2001 to examine the effect of TN:TP (total phosphorus) ratios and P-reduction on the occurrence of Microcysitis blooms. The treatments were performed with enough amounts of N but with different amounts of P in the water column and sediment. Microcystis blooms occurred in the enclosures either with an initial TN:TP <29 or TN:TP>29 where the nutrients (N, P) were high enough. Microcysitis blooms never occurred in the treatments with low P concentration in spite of the presence of sufficient N. The P-rich sediments served as an important source for the P supply in the water column, and such a process was activated greatly by the outburst of Microcystis blooms which pumped up selectively P from the sediments and thus decreased the TN:TP ratios. Therefore, the low TN:TP ratio is not a cause but rather a result of Microcystis blooms.

Eutrophication↗