Biomedical subjects

D Wen

Publications and source records attributed to D Wen.

At least 19 recordsLinked to original sources

PubMed1998

Release of the neuregulin functional polypeptide requires its cytoplasmic tail.

Based on both in vivo and in vitro studies, we have shown previously that the intracellular domain of a membrane-bound isoform of the growth factor, neuregulin, regulates proteolytic release of its extracellular domain ErbB receptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfection in COS-7 cells. These studies revealed that regulation of extracellular domain cleavage by the intracellular domain is sequence-specific and involves three distinct 30-60-residue segments. The presence of any two of these three segments is both necessary and sufficient for proteolytic processing, and resistance to proteolysis is not due to an alteration in cellular localization or transport. Evidence was also obtained that regulation of extracellular domain processing involves initial intracellular domain dimerization. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Furthermore, resistance to proteolytic processing of a construct lacking a large portion of the intracellular domain was rescued with a chimera, in which the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain interactions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms.

Animals↗

PubMed1998

Transmembrane neuregulins interact with LIM kinase 1, a cytoplasmic protein kinase implicated in development of visuospatial cognition.

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

Animals↗

PubMed1998

The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness.

The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1alpha, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.

Animals↗

PubMed1998

Identification of a palmitic acid-modified form of human Sonic hedgehog.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Animals↗

PubMed1997

ErbB kinases and NDF signaling in human prostate cancer cells.

Prostate carcinoma (PCA) is the most commonly diagnosed malignancy in American men. Our knowledge of PCA growth regulation lags behind that of other cancers, such as breast and colon carcinomas. Among receptor tyrosine kinases, the ErbB family is most frequently implicated in neoplasia. We report here the expression of ErbB family kinases and their ligands in PCA cell lines and a xenograft. While ErbB1/EGFR, ErbB2/NEU, and ErbB3 were always observed in a distinct pattern, ErbB4 was not observed. Interestingly, while TGF-alpha was expressed in the majority of PCA lines, the ligand Neu Differentiation Factor/Heregulin (NDF) was expressed only in an immortalized, non-transformed prostate epithelial line. Concomitantly, there was a significant difference in biological response to these ligands. NDF inhibited LNCaP growth and induced an epithelial-like morphological change, in contrast to TGF-alpha, which accelerated cell growth. We also performed the first comprehensive analysis of NDF signaling in a prostate line. LNCaP stimulated with NDF demonstrated crosstalk between ErbB3 and ErbB2 which did not involve ErbB1. NDF also turned on several cascades, including those of PI3-K, ERK/MAPK, mHOG/p38 and JNK/SAPK, but not those of PLCgamma or the STAT family. This signaling pattern is distinct from that of TGF-alpha. The activation of mHOG by ErbB2 or ErbB3 has not been reported, and may contribute to the unusual phenotype. PI3-K activation is characterized by the formation of a striking 'activation complex' with multiple tyrosine-phosphorylated species, including ErbB3. Our studies provide a framework in which to dissect the growth and differentiation signals of prostate cancer cells.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system.

The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3' half of alk derived from chromosome 2 is fused to the 5' portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. Here, we show molecular cloning of cDNAs for both the human and mouse ALK proteins. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in specific regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its effects on specific neurons in the nervous system.

Amino Acid Sequence↗

PubMed1997

Inhibition of lipid peroxidation.

Lipid peroxy radicals (ROO.) were detected by electron spin resonance (ESR) at low temperature after formation by addition of H2O2 into a suspension of mice lymphocytes. If lymphocytes were treated with selenomethionine (Se-Met) prior to addition of H2O2, ROO. formation was inhibited in a fashion that was dependent on Se-Met concentration. Formation of ROO. in the spleen of mice was induced by 60Co irradiation. Animals that were supplemented with Na2SeO3 prior to irradiation exhibited a lower ROO. concentration than that of nontreated animals. Based on our experiments, we have concluded that Se has an oxygen-free radical scavenging effect. This should be a protective effect against lipid peroxy radical cellular attack.

Animals↗

PubMed1997

ret receptor tyrosine kinase immunoreactivity is altered in glial cell line-derived neurotrophic factor-responsive neurons following lesions of the nigrostriatal and septohippocampal pathways.

Glial cell line-derived neurotrophic factor was initially identified as a survival factor for developing midbrain dopamine neurons (for reviews, see Refs 17 and 19). Subsequent studies have demonstrated a more wide-spread role for glial cell line-derived neurotrophic factor in the developing and adult CNS. In the adult rat brain, for instance, prior administration of glial cell line-derived neurotrophic factor protects nigrostriatal dopamine neurons from 6-hydroxydopamine-induced damage. When given several weeks after 6-hydroxydopamine injection, glial cell line-derived neurotrophic factor also restores the function of these neurons. Glial cell line-derived neurotrophic factor attenuates excitotoxin-induced cell death in the striatum and hippocampal formation and protective effects of glial cell line-derived neurotrophic factor following axotomy have been reported for spinal motor neurons and basal forebrain cholinergic neurons. These findings suggest that glial cell line-derived neurotrophic factor may be a protective/restorative agent for a diverse population of neurons and imply that it may be a useful therapeutic tool for a variety of neurodegenerative diseases including Parkinson's, Huntington's and Alzheimer's diseases. The potential receptor mediating the pleiotropic effects of glial cell line-derived neurotrophic factor has been characterized only recently as a novel glycosyl-phosphatidylinositol-linked protein, GDNFR-alpha. Because GDNFR-alpha is a cell surface receptor, an additional protein(s) was thought to be involved in the glial cell line-derived neurotrophic factor signalling cascade. The identity of the likely candidate, ret, was inferred initially from indirect evidence. Not only were there remarkable similarities in the distribution of glial cell line-derived neurotrophic factor and the proto-oncogene ret in the developing rat and mouse brain, but also in the phenotype of glial cell line-derived neurotrophic factor knockout mice and mice with ret mutations. Mice with either mutation exhibited pronounced renal and enteric abnormalities, implicating the receptor tyrosine kinase protein product of the ret proto-oncogene as the glial cell line-derived neurotrophic factor signalling protein. More conclusive evidence showing that activation of GDNFR-alpha by glial cell line-derived neurotrophic factor induces phosphorylation of ret has confirmed ret as a signalling protein for glial cell line-derived neurotrophic factor. Preliminary results showing that 6-hydroxydopamine lesions of the substantia nigra markedly reduced ret messenger RNA expression, established its localization to presumably glial cell line-derived neurotrophic factor-responsive dopamine neurons in the nigrostriatal pathway. In contrast, it is not clear whether other glial cell line-derived neurotrophic factor-responsive neurons in the CNS, such as the basal forebrain cholinergic neurons and striatal neurons, also express ret, nor is it evident whether levels of the protein are regulated by disruption of the respective pathways. The present study shows that dense networks of ret immunoreactivity are distributed throughout the nigrostriatal pathway, with lower densities of staining in other brain regions, including the septohippocampal pathway. Following extensive unilateral 6-hydroxydopamine lesions of the medial forebrain bundle, ret immunoreactivity in the substantia nigra and striatum was reduced significantly, to a similar extent as tyrosine hydroxylase immunoreactivity. In contrast, excitotoxic lesions of the striatum, achieved by intrastriatal quinolinic acid injections, resulted in increased ret staining in this brain region. In addition, marked decrements in septal ret immunoreactivity were consequent to complete transections of the fimbria-fornix.

Animals↗

PubMed1997

IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life.

We have tested the role of glial cell line-derived neurotrophic factor (GDNF) in regulating a group of putatively nociceptive dorsal root ganglion (DRG) neurons that do not express calcitonin gene-related peptide (CGRP) and that downregulate the nerve growth factor (NGF) receptor tyrosine kinase, TrkA, after birth. We show that mRNA and protein for the GDNF receptor tyrosine kinase, Ret, are expressed in the DRG in patterns that differ markedly from those of any of the neurotrophin receptors. Most strikingly, a population of small neurons initiates expression of Ret between embryonic day 15.5 and postnatal day 7.5 and maintains Ret expression into adulthood. These Ret-expressing small neurons are selectively labeled by the lectin IB4 and project to lamina IIi of the dorsal horn. Ret-expressing neurons also express the glycosyl-phosphatidyl inositol-linked (GPI-linked) GDNF binding component GDNFR-alpha and retrogradely transport 125I-GDNF, indicating the presence of a biologically active GDNF receptor complex. In vitro, GDNF supports the survival of small neurons that express Ret and bind IB4 while failing to support the survival of neurons expressing TrkA and CGRP. Together, our findings suggest that IB4-binding neurons switch from dependence on NGF in embryonic life to dependence on GDNF in postnatal life and are likely regulated by GDNF in maturity.

Aging↗

PubMed1997

Neonatal oligodendrocytes contain and secrete neuregulins in vitro.

The factors that influence the development of oligodendrocyte (OLG) progenitors into mature OLGs remain elusive. Recent evidence has suggested that neu differentiation factor (NDF), which is a member of the neuregulin family of growth factors, influences the development of glial cells, including Schwann cells, astrocytes, and OLGs. Neurons are postulated to be the source of neuregulins, because neurons closely interact with these glial cells during development. In this report, we have identified the mRNA for both isoform families of NDF in cultured neonatal (immature) OLGs. We have also demonstrated that cultured neonatal OLGs contain and secrete NDF protein. These data raise the possibility that NDF could be used in an autocrine/paracrine loop by neonatal OLGs during development for survival, proliferation, and/or differentiation.

Animals↗

PubMed1997

[The analysis of Q-Tc interval changes in treadmill exercise-induced myocardial ischemia].

The Q-Tc interval before and after the treadmill exercise test in 48 normal subjects and 59 patients with coronary heart disease were compared. The Q-Tc interval was shorter in normal group at immediate moment, 2, 4 and 6 minutes after the exercise than before. In the coronary heart disease group, the Q-Tc interval at the same time points as above was longer after exercise compared to pre-exercise. There was no significant difference of exercise-induced Q-Tc changes in silent and symptomatic myocardial ischemia.

Adult↗

PubMed1996

Cultured neonatal Schwann cells contain and secrete neuregulins.

Neuregulins have become the focus of intense research due to their putative roles in the etiology of certain cancers and to their evolving roles in PNS and CNS development. Evidence has been presented that neuregulins are present in neuronal cells where they act as mediators of neuron-glial signaling. Consistent with this view, we now report that there is a dramatic differential response of Schwann cells to the two major isoforms of neu differentiation factor (NDF alpha and NDF beta). NDF beta is a potent mitogen, whereas NDF alpha is nonmitogenic for Schwann cells. In addition, we report that Schwann cells contain endogenous NDF as well as the mRNA for both isoforms of NDF. Evidence is also presented that several isoforms of NDF are released from cultured Schwann cells. Our data suggest that in addition to functioning as a neuron-glial mediator, endogenous neuregulins may function in an autocrine/paracrine loop or in a juxtacrine- (cell-to-cell) mediated signal between individual Schwann cells or between Schwann cells and neurons.

Animals↗

PubMed1996

ErbB receptor activation, cell morphology changes, and apoptosis induced by anti-Her2 monoclonal antibodies.

A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells. These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphorylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb, mAb74, induced cell morphology changes and apoptosis.

Animals↗

PubMed1996

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

Animals↗

PubMed1996

The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure.

Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible for function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method for the location of disulfide bonds in proteins.

Actins↗

PubMed1996

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

Amino Acid Sequence↗

PubMed1996

Transformation of NIH 3T3 cells by HER3 or HER4 receptors requires the presence of HER1 or HER2.

Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF.

3T3 Cells↗

PubMed1996

Open fracture of the tibia in children.

The records of eighty-three children who had had an open fracture of the tibial metaphysis or diaphysis between January 1983 and July, 1993 were studied retrospectively. The average duration of follow-up was fourteen months (range, two to seventy-five months). There were twenty-four grade-I, forty grade-II, thirteen grade-IIIA, six grade-IIIB, and no grade-IIIC fractures, according to the classification scheme of Gustilo et al. Sixty patients (72 per cent) had sustained the fracture when they were struck by an automobile, and forty-eight patients (58 per cent) had other associated major injuries. All fractures were treated with irrigation and débridement, and antibiotics were administered parenterally for a minimum of forty-eight hours. Thirty-two patients were managed with immobilization in a cast only; forty, with transcutaneous fixation with an average of two Steinmann pins followed by immobilization in a cast; nine, with external fixation; one, with open reduction and internal fixation with two screws and two pins; and one, with delayed intramedullary nailing. Fifty-seven wounds were closed primarily (forty-four, over a Penrose drain, and thirteen, without a drain), ten were treated with delayed closure, four were allowed to heal by secondary intention, seven were covered with a soft-tissue flap, and five were treated with skin-grafting (a split-thickness skin graft was used for four, and a split-thickness and a full-thickness skin graft were used for one). The average time to union was fifteen weeks (range, five to sixty-one weeks), with the fracture healing by sixteen weeks in sixty-four patients (77 per cent). Eighteen patients (22 per cent) had delayed union, and only one patient (1 per cent) had non-union. Secondary procedures were necessary to achieve union in only two patients. Two patients had a superficial wound infection, and no patient had osteomyelitis. One patient, who had been managed with external fixation, had a pin-track infection; none of the patients who had had transcutaneous fixation had a pin-track infection. Two patients had a compartment syndrome, and two patients had a transient stretch injury of a nerve (the peroneal nerve in one and the sciatic nerve in the other). Four fractures healed with an angulatory deformity of more than 10 degrees in any plane. Five patients had overgrowth of the limb of one centimeter or more. Physeal arrest did not occur in any patient. We concluded that treatment of unstable open fractures of the tibia in children with débridement and transcutaneous fixation followed by immobilization in a cast leads to good anatomical and functional results. We prefer this technique to external fixation, which is associated with several potential complications. Loose closure of a clean open wound over a Penrose drain is effective and can be safely utilized in selected children.

Adolescent↗