A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury

Several lines of evidence suggest that neurotrophin administration may be of some therapeutic benefit in the treatment of peripheral neuropathy. However, a third of sensory neurons do not express receptors for the neurotrophins. These neurons are of small diameter and can be identified by the binding of the lectin IB4 and the expression of the enzyme thiamine monophosphatase (TMP). Here we show that these neurons express the receptor components for glial-derived neurotrophic factor (GDNF) signaling (RET, GFRalpha-1, and GFRalpha-2). In lumbar dorsal root ganglia, virtually all IB4-labeled cells express RET mRNA, and the majority of these cells (79%) also express GFRalpha-1, GFRalpha-2, or GFRalpha-1 plus GFRalpha-2. GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression. GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells and the A-fiber sprouting into lamina II of the dorsal horn. GDNF therefore may be useful in the treatment of peripheral neuropathies and may protect peripheral neurons that are refractory to neurotrophin treatment.     

GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney

A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-micron-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2 and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens and in situ hybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2 system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.     

GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons

A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific; GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration of midbrain dopaminergic neurons.     

Expression of GDNF family receptor components during development: implications in the mechanisms of interaction

Glial cell line-derived neurotrophic factor (GDNF) and a related factor, neurturin, promote survival of diverse groups of neurons. Both GDNF and neurturin signal via a two-component receptor complex that consists of a ligand-binding GDNF family receptor (GFRalpha-1 or GFRalpha-2) and the receptor protein tyrosine kinase Ret. Recently, a third receptor related to GFRalpha-1 and GFRalpha-2 has also been isolated and designated GFRalpha-3. Although much is known about the interaction among GDNF family factors, Ret, and the alpha-receptors in vitro, it remains unclear about their interactions in vivo. We show here by in situ hybridization that Ret and the alpha-receptors may be colocalized in the same tissues or expressed separately in projecting and target tissues, respectively, indicating that two distinct modes of interaction between Ret and the alpha-receptors exist in vivo. First, Ret may interact with the alpha-receptors expressed in the same cells (termed interaction "in cis") in many tissues and cell populations that respond to GDNF and/or neurturin, such as the substantia nigra, dorsal root ganglia, spinal cord motoneurons, kidney, and intestine. Second, Ret may interact with the alpha-receptors localized in the target neurons (termed interaction "in trans"). In addition, we present evidence in vitro that GFRalpha-1 mediates Ret activation by GDNF in trans. These observations suggest that there are multiple mechanisms regulating the interaction between Ret and the alpha-receptors that mediates the effects of GDNF family trophic factors on the survival and differentiation of cells and on neuron-target interactions in the nervous system.     

CNS glia are targets for GDNF and neurturin

Carboplatin is one of the most common drugs used for radiochemotherapy of cancer. However, the best way to combine the drug with fractionated radiotherapy has not been established. In the present study the authors investigated which maximum tolerated daily bolus dose of carboplatin would allow safe radiopotentiation for 10 consecutive radiotherapy days, the scheme being repeated twice during the 6 weeks that a conventional radiotherapy scheme lasts. Seventy-two patients with lung or pelvis malignancies were included in a dose escalation study. Twenty-four patients comprised the first baseline cohort and were treated with radiotherapy alone. The daily dose of carboplatin was escalated starting from 38 mg/m2 daily (for 10 days) and increasing by 7 mg/m2 per day. Six patients were to be included in each cohort. All 12 patients treated at the 38 mg/m2 and 45 mg/m2 dose level completed two cycles of 10-day carboplatin treatment with no grade III-IV toxicity. Granulocyte colony-stimulating factor effectively averted the incidence of neutropenia and allowed the administration of the second carboplatin 10-day cycle in five of six patients at the 52 mg/m2 daily dose level. Platelet grade III-IV toxicity was observed in all 12 patients (six supported with granulocyte colony-stimulating factor and six with granulocyte colony-stimulating factor and recombinant human erythropoietin) treated at the 59 mg/m2 daily dose level and none of them received the second cycle of chemotherapy. Twelve patients were treated at the same dose level using amifostine 500 mg before carboplatin infusion. Two patients interrupted chemotherapy because of severe nausea and vomiting. Nine of 10 who accomplished the 10-day treatment had platelet levels more than 90,000/microl on day 28 and completed the second 10-day cycle without severe toxicity. Acute radiation toxicity did not increase in the carboplatin cohorts. In this study the authors established a high-dose fractionated carboplatin schedule that can be safely administered during radical radiotherapy.     

Characterization of glucuronic acid conjugates of a novel angiotensin receptor antagonist

Nanogram quantities of glucuronic acid conjugates of GR117289 in rat and dog bile have been analysed by semi-microbore high-performance liquid chromatography (HPLC)/ionspray mass spectrometry with on-line UV diode array detection. The determination of drug metabolites in bile has often proved problematical due to the large number of endogenous components in this biological matrix, in particular the bile acids. Semi-microbore HPLC is useful for concentrating small quantities of material and, in combination with an on-line diode array detector, for distinguishing between drug related and endogenous components. A novel angiotensin II receptor antagonist, GR117289, had proved difficult to analyse by thermospray mass spectrometry because of its thermal lability. The use of the less thermally dependent technique of ionspray mass spectrometry allowed the characterization of nanogram quantities of glucuronic acid metabolites of GR117289 in bile.     

Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a Kd of about 35 microg/ml, similar to the Kd for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a "docking receptor" for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Macrosegregation in a multicomponent low alloy steel

Macrosegregation theory is extended to predict the formation of channel-type segregation for multicomponent systems. Specifically, calculations are carried out for 0.7 pct C steel, by considering heat, mass and momentum transport in the mushy zone. In the model used for calculations the momentum transport equation and the energy equation were solved simultaneously. It is confirmed, by comparing calculated results with experimental results, that this model successfully predicts the occurrence of channel-type segregation. This analysis is also more rigorous than previous works on macrosegregation because previous analyses were done by solving for convection in the mushy zone with an “uncoupled” temperature field. Using the model, the effects of adjusting the compositions of silicon and molybdenum in steel were quantitatively evaluated in order to show how channel-type segregates can be avoided by adjusting alloy composition. A method of optimizing composition to minimize segregation is presented. It is recommended that this methodology be applied to alloy design so that ingots of alloys amenable to commercial practice can be obtained readily with a minimum amount of “trial-and-error” development work and expense. Formerly Research Affiliate, Department of Materials Science and Engineering, Massachusetts Institute of Technology AR, was Visiting Scientist, Department of Materials Science and Engineering, Massachusetts Institute of Technology     

Characterization of the detergent insolubility of the T cell receptor for antigen

Aggregation of cell surface receptors plays an important role in signal transduction in many receptor systems. In the T cell receptor (TCR), as in many other cell surface receptors, this aggregation results in insolubility in certain nonionic detergents. We have characterized this insolubility for TCR, and we show it is not preexisting in HPB-ALL cells but increases with increasing TCR aggregation. It is not likely to be due to a direct interaction with cellular cytoskeletal elements, as it is not affected by inhibitors of actin or tubulin polymerization. It may be due to interaction with detergent-resistant membrane domains that have been found in various cell types and contain tyrosine kinases, the earliest known participants in TCR signal transduction. This aggregation-dependent insolubility occurs as rapidly as the anti-TCR antibody binds, so the kinetics are consistent with an involvement in signal transduction. It is not, however, dependent on signal transduction, as inhibitors of tyrosine kinases do not inhibit the insolubility. Insolubility is also enhanced by preaggregation of CD4, an important T cell surface molecule which also associates with the tyrosine kinase p56lck.     

Characterization and function of the receptor for IGF-I in human preosteoclastic cells

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.     

Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells

A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of approximately 5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly alpha-helices (50%) and beta-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.