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Biomedical subjects

E Li

Publications and source records attributed to E Li.

At least 73 records · Page 4Linked to original sources
PubMed1999

The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

ActRIA (or ALK2), one of the type I receptors of the transforming growth factor-beta (TGF-beta) superfamily, can bind both activin and bone morphogenetic proteins (BMPs) in conjunction with the activin and BMP type II receptors, respectively. In mice, ActRIA is expressed primarily in the extraembryonic visceral endoderm before gastrulation and later in both embryonic and extraembryonic cells during gastrulation. To elucidate its function in mouse development, we disrupted the transmembrane domain of ActRIA by gene targeting. We showed that embryos homozygous for the mutation were arrested at the early gastrulation stage, displaying abnormal visceral endoderm morphology and severe disruption of mesoderm formation. To determine in which germ layer ActRIA functions during gastrulation, we performed reciprocal chimera analyses. (1) Homozygous mutant ES cells injected into wild-type blastocysts were able to contribute to all three definitive germ layers in chimeric embryos. However, a high contribution of mutant ES cells in chimeras disrupted normal development at the early somite stage. (2) Consistent with ActRIA expression in the extraembryonic cells, wild-type ES cells failed to rescue the gastrulation defect in chimeras in which the extraembryonic ectoderm and visceral endoderm were derived from homozygous mutant blastocysts. Furthermore, expression of HNF4, a key visceral endoderm-specific transcription regulatory factor, was significantly reduced in the mutant embryos. Together, our results indicate that ActRIA in extraembryonic cells plays a major role in early gastrulation, whereas ActRIA function is also required in embryonic tissues during later development in mice.

Activin Receptors, Type I↗
PubMed1999

Highly metastatic human prostate cancer growing within the prostate of athymic mice overexpresses vascular endothelial growth factor.

Angiogenesis is essential for tumor progression and metastasis. It is mediated by the release of angiogenic factors by the tumor or host. We analyzed the expression of angiogenic factors by the prostate cancer cell line LNCaP and two derived variants, in vitro and in vivo, to determine whether metastatic cell lines express higher levels of these factors. The production of three angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin 8 (IL-8), by LNCaP and its variants, LNCaP-LN3 (highly metastatic) and LNCaP-Pro5 (slightly metastatic), was measured by ELISA. VEGF, bFGF, and IL-8 mRNA expression was determined in vitro by Northern blot analysis. VEGF mRNA expression was determined in vivo by in situ hybridization. VEGF and flk-1 protein expression and microvessel density of LNCaP cell tumors were quantified by immunohistochemistry. In vitro, VEGF production by LNCaP-LN3 (3.15+/-0.04 pg/ml/10(3) cells) was significantly higher than those of both LNCaP (2.38+/-0.34 pg/ml/10(3) cells) and LNCaP-Pro5 (1.67+/-0.37 pg/ml/10(3) cells; P = 0.049 and 0.001, respectively). None of the three cell lines produced detectable levels of bFGF or IL-8 in vitro. In vivo, LNCaP-LN3 tumors exhibited higher levels of VEGF mRNA and protein (152.2+/-28.5 and 200.5+/-28.3) and of flk-1 protein (156.5+/-20.6) and had higher microvessel density (16.4+/-4.2) than either LNCaP tumors (89+/-17.5, 173.3+/-23.0, 124.6+/-21.6, and 12.4+/-3.5, respectively) or LNCaP-Pro5 tumors (63+/-14.7, 141.2+/-38.1, 126.1+/-20, and 5.8+/-2.2, respectively). In conclusion, metastatic human prostate cancer cells exhibited enhanced VEGF production and tumor vascularity compared with prostate cancer cells of lower metastatic potential. Thus, VEGF may play an important role in prostate cancer metastasis.

Animals↗
PubMed1999

Structure and function of cytoplasmic retinoid binding proteins.

We examined the ligand protein interactions of two highly homologous cellular retinol binding proteins, CRBP and CRBP-II, and two highly homologous cellular retinoic acid binding proteins, CRABP-I and CRABP-II. While the crystal structures of all four have been determined, nuclear magnetic resonance studies provide a means for observing dynamic aspects of ligand protein interactions of these proteins in solution. The cellular functions of these proteins are less well understood. We have modeled retinoid flux between cytoplasmic retinoid proteins and model membranes and with nuclear receptors. Based on our in vitro studies, we propose that certain retinoids may indirectly influence retinoid signaling by displacing endogenous retinoids from the cytoplasmic proteins to the nuclear receptors.

Cytoplasm↗
PubMed1998

The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation.

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

Animals↗
PubMed1998

Smad2 role in mesoderm formation, left-right patterning and craniofacial development.

Signalling by the transforming growth factor-beta (TGF-beta) superfamily of proteins depends on the phosphorylation and activation of SMAD proteins by heteromeric complexes of ligand-specific type I and type II receptors with serine/threonine-kinase activity. The vertebrate SMAD family includes at least nine members, of which Smad2 has been shown to mediate signalling by activin and TGF-beta. In Xenopus, Smad2 can induce dorsal mesoderm, mimicking Vg-1, activin and nodal. Here we investigate the function of Smad2 in mammalian development by generating two independent Smad2 mutant alleles in mice by gene targeting. We show that homozygous mutant embryos fail to form an organized egg cylinder and lack mesoderm, like mutant mice lacking nodal or ActRIB, the gene encoding the activin type-I receptor. About 20 per cent of Smad2 heterozygous embryos have severe gastrulation defects and lack mandibles or eyes, indicating that the gene dosage of Smad2 is critical for signalling. Mice trans-heterozygous for both Smad2 and nodal mutations display a range of phenotypes, including gastrulation defects, complex craniofacial abnormalities such as cyclopia, and defects in left-right patterning, indicating that Smad2 may mediate nodal signalling in these developmental processes. Our results show that Smad2 function is essential for early development and for several patterning processes in mice.

Abnormalities, Multiple↗
PubMed1998

Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells.

We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase. In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast. Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase. However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells. We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells. Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells.

Amino Acid Sequence↗
PubMed1998

Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300.

The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.

Animals↗
PubMed1998

Defects in regulation of apoptosis in caspase-2-deficient mice.

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

Amyotrophic Lateral Sclerosis↗
PubMed1998

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse.

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB-/- embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB-/- ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB-/- cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB-/- blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation.

Activin Receptors, Type I↗
PubMed1998

Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE.

We report here the inactivation of a member of the Ice/Ced-3 (caspase) family of cell death genes, casp-11, by gene targeting. Like Ice-deficient mice, casp-11 mutant mice are resistant to endotoxic shock induced by lipopolysaccharide. Production of both IL-1alpha and IL-1beta after lipopolysaccharide stimulation, a crucial event during septic shock and an indication of ICE activation, is blocked in casp-11 mutant mice. casp-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of ICE. Furthermore, we found that pro-caspase-11 physically interacts with pro-ICE in cells, and the expression of casp-11 is essential for activation of ICE. Our data suggest that caspase-11 is a component of ICE complex and is required for the activation of ICE.

Animals↗
PubMed1998

Phosphorylation of the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function.

The pheromone-responsive Gbeta subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310-346, ste4delta310-346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho-) alleles of STE4: ste4-T320A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4delta310-346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive Galpha of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310-346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.

Amino Acid Sequence↗
PubMed1998

Epithelial cell-initiated inflammation plays a crucial role in early tissue damage in amebic infection of human intestine.

BACKGROUND & AIMS: Entamoeba histolytica infection of the intestine can induce severe gut inflammation. The aims of this study were to assess the role of the host inflammatory response in the tissue damage observed with amebiasis and the role of the intestinal epithelial cell in initiating that response. METHODS: E. histolytica infection was established in human intestinal xenografts in severe combined immunodeficient (SCID-HU-INT) mice. Human intestinal epithelial cell inflammatory responses to amebic infection were inhibited by the intraluminal administration of an antisense oligonucleotide to the human p65 subunit of nuclear factor kappaB, and the role of neutrophils in tissue damage observed with amebiasis was studied by depleting neutrophils from SCID-HU-INT mice. RESULTS: Administration of the antisense oligonucleotide blocked the production of human interleukin 1beta and interleukin 8 by intestinal epithelial cells and inhibited neutrophil influx into the E. histolytica-infected intestinal xenografts. Inhibition of the gut inflammatory response by the antisense oligonucleotide or the depletion of neutrophils from SCID-HU-INT mice blocked the increase in intestinal permeability observed with amebic infection. CONCLUSIONS: Intestinal epithelial cells initiate an inflammatory response with resulting neutrophil-mediated tissue damage in response to E. histolytica infection; this inflammatory cascade can be blocked by inhibiting the transcription of genes regulated by nuclear factor kappaB.

Amebiasis↗
PubMed1998

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment.

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.

Adaptation, Physiological↗
PubMed1998

Disease-modifying antirheumatic drugs.

Disease-modifying antirheumatic drug (DMARD) therapy is now clearly accepted as the primary treatment for rheumatoid arthritis, with an increasing emphasis on use of combination therapy. Data on combination therapy have highlighted the difficulties in performing these studies and the large number of patients required to produce meaningful results. Combination studies have focused on use of rapidly decreasing high-dose steroids as a part of the combination and emphasize the importance of using patients with early rheumatoid arthritis. Even with relatively aggressive use of DMARDs, the majority of patients develop erosions. Adverse reactions to DMARDs continue to concern clinicians, although evaluation of the frequency of these events has led to a reappraisal of previously accepted monitoring strategies in some cases. For example, it may not be cost-effective to subject patients on antimalarials to regular review by an ophthalmologist because of the low frequency of serious eye defects. Studies have also identified risk factors for the development of pulmonary toxicity in association with methotrexate. That DMARDs are effective in treating rheumatoid arthritis is beyond question-just how effective they are and what combinations of DMARDs will show improved efficacy will provide data for the next annual review.

Antimalarials↗
PubMed1998

Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases.

Adenovirus (Ad) endocytosis via alphav integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

Actins↗
PubMed1998

Adenovirus endocytosis via alpha(v) integrins requires phosphoinositide-3-OH kinase.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

Adenoviruses, Human↗