Biomedical subjects

R H Xu

Publications and source records attributed to R H Xu.

At least 19 recordsLinked to original sources

PubMed2004

Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabine.

B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.

Antineoplastic Agents↗

PubMed2000

Maternal cold inducible RNA binding protein is required for embryonic kidney formation in Xenopus laevis.

We cloned a major isoform of Xenopus homologue of cold inducible RNA binding protein (CIRP), XCIRP-1. XCIRP-1 was neither cold inducible nor essential for cell division during early embryonic development. Suppression of XCIRP-1 dose dependently produced tailbuds with deformations of the brain and internal organs. The defects were XCIRP-1 specific as they could be rescued by sense transcript. Suppression of XCIRP-1 also disrupted the morphogenetic migration of the C3 blastomeres (lineaged to become the embryonic kidney, the pronephros). In animal cap explants, depletion of XCIRP-1 inhibited activin/retinoic acid induced expressions of pronephros related Xlim-1 and WT1 genes. These results suggest that XCIRP-1 is required for the specification and morphogenetic lineage migration of the pronephros.

Abnormalities, Multiple↗

PubMed2000

Characterization of the functionally related sites in the neural inducing gene noggin.

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.

Animals↗

PubMed2000

Histone acetylation is a checkpoint in FGF-stimulated mesoderm induction.

We have previously demonstrated that the transcription factor, AP-1 (c-jun/c-fos heterodimer), mediates fibroblast growth factor (FGF) signaling during mesoderm induction in Xenopus embryo. In the present studies, we show that histone acetylation is involved in FGF-mediated signaling leading to mesoderm induction. Histone acetylation is a dynamic process regulated by the activities of two histone-modifying enzymes, the histone acetyltransferase(s) and histone deacetylase(s) (HDACs). We found that basal and FGF-regulated activator protein 1 (AP-1) activity in Xenopus embryo is markedly reduced by treatment of trichostatin A (TSA), a specific inhibitor of HDAC. However, activity of another transcription factor, NFkappaB, is enhanced by TSA treatment. AP-1-mediated mesoderm induction in the animal caps is dramatically suppressed by TSA at a dose-dependent manner. This suppression can be rescued by ectopic expression of HDAC3 at early stage. Finally, we found that histone acetylation in animal caps is inhibited by FGF whereas enhanced by TSA (as a control). Therefore, we propose that histone acetylation is a checkpoint for transduction of the FGF/AP-1 signals to induce mesoderm. Published 2000 Wiley-Liss, Inc.

Acetylation↗

PubMed1999

Functional analysis of human Smad1: role of the amino-terminal domain.

The signals originating from transforming growth factor beta/activin/bone morphogenetic proteins (BMPs) are transduced by a set of evolutionarily conserved family of Smad proteins which, upon activation, directly translocate to the nucleus where they may activate transcription. Smad proteins of different species contain conserved amino- (N) and carboxy- (C) terminal domains separated by a proline-rich linker. Human, Drosophila, and Xenopus Smad1 all have been shown to mediate the biological effects of BMP-4 in Xenopus embryos. We have investigated the functional domains of human Smad1 (hSmad1) using the Xenopus embryo system. Dorsal injection of hSmad1 RNA into the 4-cell-stage embryos results in embryonic ventralization. Since the C-terminus of Smads has been shown to mediate the transcriptional activity, whereas this activity is masked by the presence of the N-terminus, we tested the effect of a hSmad1 construct lacking the C-terminal domain [hSmad1(N)] in the Xenopus embryo system. Surprisingly, we found that hSmad1(N) not only synergizes with hSmad1 in embryonic ventralization, but induces ventralization by itself. Ectopic expression of a dominant negative BMP receptor (DN-BR) as well as neural inducers noggin and chordin induce neurogenesis in the animal cap, which is inhibited by co-expression of either hSmad1 or hSmad1(N). Ventral expression of DN-BR induces formation of a second body axis at tailbud stage, which is also prevented by hSmad1 and hSmad1(N). It has recently been reported that calmodulin interacts with the N-terminal domain of Smad proteins. We demonstrate that the ventralizing activity of hSmad1 and hSmad1(N) is markedly inhibited by calmodulin. Thus, calmodulin acts as a Smad1 inhibitor. A model is proposed to accomodate these findings.

Animals↗

PubMed1999

Opposite effects of FGF and BMP-4 on embryonic blood formation: roles of PV.1 and GATA-2.

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. Here we address the role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo. We report that ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, we examined how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. We observed that eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which we demonstrate to inhibit blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of each other. Thus, BMP-4 induces two transcription factors which have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage.

Animals↗

PubMed1998

Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP-4 and its promoter.

Recent experiments in the Xenopus embryo suggest that proper regulation of BMP-4 signaling is critical to the dorsal ventral specification of both mesoderm and ectoderm. Regulation of BMP-4 signaling is known to occur extracellularly by direct binding with chordin, noggin, and follistatin, and intracellularly through the antagonistic signal interaction with dorsalizing TGF-beta family member activin. However, tight repressional regulation of BMP transcription may also be required to sustain the dorsal and neural status of the induced cells. Here we demonstrate that the dominant negative mutant of the BMP receptor (DN-BR) or the BMP-4 antagonizers, chordin and noggin, negatively regulate BMP-4 transcription in animal cap explants. We suggest that repression of BMP-4 transcription is important in the maintenance of dorsal fate and that continuous input of BMP-4 signaling is required to sustain the expression of BMP-4 transcription in the maintenance of epidermal/ventral fate. Consistent with this postulation, we found that the promoter region of the isolated BMP-4 genomic DNA includes several consensus binding sites for transcriptional regulators functioning under BMP-4 signaling such as GATA binding and ventralizing homeobox genes. In a functional assay we found that the GATA binding and ventral homeobox proteins can positively modulate BMP-4 promoter activity. We also observed that DN-BR decreases BMP-4 promoter activity. This was likely due to a repression of the above-mentioned transcription factors. The significance of these observations to embryonic patterning is discussed.

Animals↗

PubMed1998

Xenopus CRMP-2 is an early response gene to neural induction.

A neural specific protein, CRMP-2 (for Collapsin Response Mediator Protein-2), is considered to mediate collapsin-induced growth cone collapse during neural development. We have isolated the Xenopus homologue of the CRMP-2 (XCRMP-2) cDNA and studied the expression of XCRMP-2 mRNA and protein during neural induction. Induction of XCRMP-2 mRNA and protein expression, like N-CAM, occurred at the midgastrula stage and increased through early neural developmental stages. Whole mount in situ hybridization demonstrated that expression of XCRMP-2 mRNA was localized in neural tissues such as the neural plate and tube at early stages, while its expression in the brain, spinal cord, and eyes was observed at later stages. Immunostaining of Xenopus embryos with the antibody against CRMP-2 also showed that the protein was specifically expressed in the neural tissues at early stages. XCRMP-2 expression was induced by neural inducers such as noggin and chordin which antagonize a neural inhibitor, BMP4. A dominant negative BMP receptor also induced XCRMP-2 expression, suggesting that transcription of XCRMP-2 gene was negatively regulated by the BMP4 signaling. These results indicate that expression of XCRMP-2 is an early response marking neural commitment, and that transcriptional control of XCRMP-2 gene, is one of the targets of BMP4 signaling.

Amino Acid Sequence↗

PubMed1998

The immunomodulator AS101 restores T(H1) type of response suppressed by Babesia rodhaini in BALB/c mice.

The immunomodulator AS101 has been previously shown to confer protection upon BALB/c mice infected with the intraerythrocytic parasite Babesia rodhaini (B. rodhaini). The present study focuses on the effect of AS101 administration on the acute phase of babesial infection where T helper cell subset patterns-TH1/TH2-were assessed in heavily infected mice. Secretion of cytokines of the TH1 subset (IL-2, IFN-gamma, IL-12) and of the TH2 subset (IL-10, IL-4) as well as TGF-beta was measured following the administration of AS101 2 weeks before parasite infection. Our results demonstrate that the parasites suppress IL-2 protein and IL-12 mRNA and that AS101 upregulates their secretion: IL-2, 8 u/ml vs 34 u/ml, respectively; IFN-gamma protein, 2370 pg/ml vs 4777 pg/ml, respectively. Conversely, babesial infection results in the upregulation of IL-10 and IL-4 proteins and TGF-beta transcripts, whereas AS101 downregulates their production: IL-10, 1800 pg/ml vs 360 pg/ml, respectively; IL-4, 58.3 pg/ml vs 24.5 pg/ml, respectively. A possible escape mechanism induced by B. rodhaini is suggested, starting with IL-10 inhibition of macrophage activities leading to a suppression of the TH1 response and of IL-2 in particular. It is therefore possible that AS101 may protect infected mice by activating cellular-mediated immunity and concurrently balancing the TH subset responses. It is suggested that AS101 may be effective as an antiparasitic drug.

Adjuvants, Immunologic↗

PubMed1998

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.

Animals↗

PubMed1998

Mesoderm induction by heterodimeric AP-1 (c-Jun and c-Fos) and its involvement in mesoderm formation through the embryonic fibroblast growth factor/Xbra autocatalytic loop during the early development of Xenopus embryos.

We have previously demonstrated the involvement of AP-1/Jun in fibroblast growth factor (FGF) signaling by loss-of-function assay (Dong, Z., Xu, R.-H., Kim, J., Zhan, S.-N., Ma, W.-Y., Colburn, N. H., and Kung, H. (1996) J. Biol. Chem. 271, 9942-9946). Further investigations by gain-of-function are reported in this study. AP-1 transactivation activity was increased by the treatment of animal cap explants with FGF. Ectopic overexpression of two components of AP-1 (c-jun and c-fos together, but not alone) produced posteriorized embryos and induced mesoderm formation in animal cap explants, indicating that both AP-1 heterodimers are required for mesoderm induction. Since Ras/AP-1 functions downstream of FGF signaling, we then tested the involvement of Ras/AP-1 in mesoderm maintenance mediated by embryonic FGF/Xbra using dominant-negative mutants. Mesoderm maintenance mediated by embryonic FGF/Xbra was blocked by dominant-negative mutants of Ras/AP-1, and AP-1 enhanced the expression of Xbra. Further studies demonstrated the inhibition of Ras/AP-1-mediated mesoderm formation by dominant-negative mutants of the FGF receptor and Xbra. These results indicate that Ras/AP-1 and FGF/Xbra signals are involved in the mesoderm maintenance machinery and mesoderm formation through the synergistic action of the diversified signal pathways derived from the FGF/Xbra autocatalytic loop.

Animals↗

PubMed1997

The homeobox gene PV.1 mediates specification of the prospective neural ectoderm in Xenopus embryos.

Bone morphogenetic protein 4 (BMP4), a member of the TGF beta superfamily, has been implicated in the dorsoventral specification of both mesoderm and ectoderm. High levels of BMP4 signaling appear to specify ventral lineages, while lower levels are causally associated with the development of dorsal lineages. We have previously identified a homeobox-containing transcription factor (PV. 1) which is a likely mediator of the ventralizing effects of BMP4 in the mesoderm. Here we provide evidence that PV.1 also functions downstream of BMP4 in the patterning of ectoderm, specifying epidermal and suppressing neural gene expression. PV.1 is expressed in the prospective neuroectoderm at the time of ectodermal fate determination. BMP4 and xSmad1 (a downstream effector of BMP4) induce PV.1 in uncommitted ectoderm and the dominant negative form of the BMP4 receptor (DN-BR) blocks PV.1 expression. In animal pole explants PV.1 counteracts the neuralizing effects of chordin and the DN-BR and restores them to their original epidermal fate. To address the physiological significance of these observations we employed an animal cap transplantation system and demonstrated that overexpression of PV.1 in the prospective neuroectoderm specifically blocks neurogenesis in intact embryos. Thus, PV.1 plays an important role in the ventralization of both mesoderm and ectoderm. We have previously shown that PV.1 is also preferentially expressed in the ventral endoderm, suggesting that this transcription factor may be involved in the ventralization of all three germ layers.

Amino Acid Sequence↗

PubMed1997

Studies on the role of fibroblast growth factor signaling in neurogenesis using conjugated/aged animal caps and dorsal ectoderm-grafted embryos.

Basic fibroblast growth factor (bFGF) has been shown to induce neural fate in dissociated animal cap (AC) cells or in AC explants cultured in low calcium and magnesium concentrations. However, long-term disclosure of the cap may cause diffusion of the secreted molecule bone morphogenetic protein 4 (BMP-4), a neural inhibitor present in the AC. This may contribute to the subsequent neurogenesis induced by bFGF. Here we used conjugated and aged blastula AC to avoid diffusion of endogenous molecules from the AC. Unlike noggin, bFGF failed to induce neural tissue in this system. However, it enhanced neuralization elicited by a dominant negative BMP receptor (DN-BR) that inhibits the BMP-4 signaling. Posterior neural markers were turned on by bFGF in AC expressing DN-BR or chordin. Blocking the endogenous FGF signal with a dominant negative FGF receptor (XFD) mainly inhibited development of posterior neural tissue in neuralized ACs. These in vitro studies were confirmed in vivo in embryos grafted with XFD-expressing ACs in the place of neuroectoderm. Expression of some regional neural markers was inhibited, although markers for muscle and posterior notochord were still detectable in the grafted embryos, suggesting that XFD specifically affected neurogenesis but not the dorsal mesoderm. The use of these in vitro and in vivo model systems provides new evidence that FGF, although unable to initiate neurogenesis on its own, is required for neural induction as well as for posteriorization.

Animals↗

PubMed1997

Lead-induced developmental changes in AP-1 DNA binding in rat brain.

Exposure to lead during ontogeny is detrimental to the growth and development of the brain. Morphological abnormalities occur in the developing brain, which are manifested as mental retardation and other neurological disorders. Despite extensive research, the biochemical mechanism for neurological effects of lead has not been established but appears to be at the level of the genome since aberrant expression of developmentally-important genes has been reported. Basal levels of activator protein 1 (AP-1) transcription factor DNA binding are elevated in the rat brain during the early postnatal period. The AP-1 DNA binding complex is composed of a Jun:cAMP responsive element binding protein dimer, which appears to modulate expression of developmentally-important genes that contain AP-1 binding sites in their promoter. Brain regions from perinatally lead-exposed rats were examined on postnatal days 3, 9 and 15 for DNA binding activity to the AP-1 DNA sequence. AP-1 DNA binding activities were significantly increased on postnatal day 3 in hippocampus and cortex from lead-treated rats with no other area (frontal lobe or brainstem) or timepoint showing significant changes. Since no increases were detected in the level of Jun protein which is a component of the AP-1 binding complex, post-translational modification may be involved in enhancing DNA binding activity. By altering levels of AP-1 DNA binding to the promoter regions, lead exposure may be changing the levels of mRNA synthesis of developmentally important genes.

Animals↗

PubMed1997

Differential regulation of neurogenesis by the two Xenopus GATA-1 genes.

Previously, we have shown that the ventralizing factor bone morphogenetic protein 4 (BMP-4) can inhibit Xenopus neurogenesis. The erythroid transcription factor GATA-1 functions downstream of the BMP-4 signaling pathway and mediates BMP-4-induced erythropoiesis. We have found that similar to BMP-4, GATA-1b inhibits neuralization of Xenopus animal cap (AC) cells. The neural inhibition is not seen with GATA-1a, although both GATA-1a and GATA-1b RNAs are translated at the same efficiency and induce globin expression equally in AC cells. GATA-1b RNA injection into AC cells neither induces expression of Xbra (a general mesoderm marker) nor affects expression of XK81 (epidermal keratin) or BMP-4 and Xvent-1 (two ventral markers). These data suggest that GATA-1b retains the epidermal fate of the AC. Intact GATA-1b protein is required for both inhibition of neurogenesis and induction of globin expression. Our findings indicate that GATA-1b can function in ectoderm to specifically regulate neural inducing mechanisms, apparently related to the expression of chordin, a neuralizing gene. Furthermore, tadpole stage embryos injected with GATA-1b are devoid of all dorsoanterior structures including neural tissue. This report provides evidence that the two transcription factors, derived from a recent genome duplication, share a common biological activity (stimulation of erythropoiesis) while also exhibiting a distinct function (inhibition of neurogenesis).

Animals↗

PubMed1997

[Prevention and treatment of seasonal asthmatic patients by combined invigorating kidney for preventing asthma tablets and beclomethasone dipropinate].

OBJECTIVE: To evaluate the efficacy of combined Invigorating Kidney for Preventing Asthma (IKPA) tablets and beclomethasone dipropinate (BDP). METHODS: Fifty seven seasonal asthmatic patients were studied. They were randomly assigned to either of the two groups: the treated group (n = 32) treated with the IKPA tablets and inhalated BDP, the control group (n = 25) inhalated BDP singly. Measurements of serum eosinophil cationic protein (ECP), soluble IL-2 receptor (SIL-2), total eosinophil counts, spirometry and methacholine challenge were performed before and after treatment. RESULTS: After 3 months' treatment and 6 months' observation the authors observed a significant improvement in both groups. The treated group had better clinical efficacy and the ECP level had reduced significantly, FEV1 increased greatly than the control group. CONCLUSIONS: The two drugs influenced the function of eosinophils and and T lymphocytes and this might contribute to the efficacy.

Adolescent↗

PubMed1996

The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis.

Vertebrate embryonic blood formation is regulated by factors that participate in dorsal-ventral patterning and mesoderm induction. The GATA-binding transcription factors are required for normal hematopoiesis and are expressed during gastrulation when ventral mesoderm (VM) is induced to form blood. Based on the recent demonstration that bone morphogenetic protein (BMP-4) is a potent ventralizing factor and inducer of hematopoietic tissue, we hypothesized that GATA-2 could be induced or activated by BMP-4. Here we demonstrate that BMP-4 can stimulate GATA-2 expression, and that expression of a dominant negative BMP-4 receptor can suppress GATA-2 induction by BMP-4 in ventral mesoderm. Over-expression of GATA-2 in ventral mesoderm leads to increased globin production and forced expression of GATA-2 in primitive ectoderm adjacent to ventral mesoderm also stimulates globin expression. Our results suggest that BMP-4 and GATA-2 can function in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation during embryogenesis.

Animals↗

PubMed1996

AP-1/jun is required for early Xenopus development and mediates mesoderm induction by fibroblast growth factor but not by activin.

In Xenopus, normal mesoderm formation depends on signaling through the fibroblast growth factor (FGF) tyrosine kinase receptor. An important signaling pathway from receptor tyrosine kinases involves Ras/Raf/MAP kinase. However, the downstream pathway that occurs in the nucleus to finally trigger gene expression for mesoderm formation remains unknown. We report here that a high level of activator protein-1 (AP-1)-dependent transcriptional activity is detected during the early development of Xenopus embryos. Injection of a dominant negative mutant jun (DNM-jun or TAM67) RNA into the two-cell stage embryos inhibited endogenous AP-1 activity and blocked normal embryonic development with severe posterior truncation in tadpoles. The inhibition of AP-1 activity and the phenotypic change induced by TAM67 was rescued by co-injection of wild-type c-jun RNA, but not by the control beta-galactosidase RNA. The FGF-stimulated mesoderm induction was markedly inhibited in animal cap explants from the embryos injected with TAM67. Activin induction of mesoderm, on the other hand, was normal in the embryos injected with TAM67 RNA. These findings suggest that AP-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and the AP-1/Jun is a key signaling molecule in the development of posterior structure.

Activins↗