Expression of neurotrophins and their receptors in human bone marrow.
(9/15103)
The expression of neurotrophins and their receptors, the low-affinity nerve growth factor receptor (p75LNGFR) and the Trk receptors (TrkA, TrkB, and TrkC), was investigated in human bone marrow from 16 weeks fetal age to adulthood. Using reverse transcription-polymerase chain reaction, all transcripts encoding for catalytic and truncated human TrkB or TrkC receptors were detected together with trkAI transcripts, whereas trkAII transcripts were found only in control nerve tissues. Transcripts for the homologue of the rat truncated TrkC(ic113) receptor were identified for the first time in human tissue. Stromal adventitial reticular cells were found immunoreactive for all neutrophin receptors. In contrast, hematopoietic cell types were not immunoreactive for p75LNGFR but showed immunoreactivity for one or several Trk receptors. TrkA immunoreactivity was found in immature erythroblasts. Catalytic TrkB immunoreactivity was observed in eosinophilic metamyelocytes and polymorphonuclear cells. Truncated TrkB immunoreactivity was found in erythroblasts and megacaryocytes. Immunoreactivity for both catalytic and truncated TrkC receptor was observed in promyelocytes, myelocytes, some polymorphonuclear cells and megacaryocytes. Neutrophin transcript levels appeared higher at fetal than at adult stages, no variation in Trk family transcript levels was observed. The local expression of neurotrophin genes suggests a wide range of paracrine and/or autocrine mode of action through their corresponding receptors within the bone marrow. (+info)
Embryonal feather growth in the chicken.
(10/15103)
Prenatal feather growth development in the chicken was studied in 7 body regions in HH stages 27-45, using direct measurements, specific histological and immunohistochemical methods, and scanning electron microscopy. The results from measurements of absolute length values, and, particularly, growth rate development in each HH stage revealed a distinct phase of most intensive growth in HH stage 40-41, which was preceded by feather follicle insertion and accompanied by the occurrence of alpha-keratins in barbule cells. Specific regional evaluation demonstrated that growth in the feather follicles of abdominal skin generally showed the slowest progression from absolute values and that in the feather filaments of the developing wings the most rapid progression occurred during HH stage 40-41 from growth rate values. (+info)
Expression and function of leptin receptor isoforms in myeloid leukemia and myelodysplastic syndromes: proliferative and anti-apoptotic activities.
(11/15103)
The receptor for the gene product of the obesity gene, leptin, was recently reported to be expressed on murine and human hematopoietic progenitor cells. Therefore, we studied the expression of the leptin receptor, OB-R, in normal myeloid precursors, human leukemia cell lines, and primary leukemic cells using reverse-transcriptase polymerase chain reaction. In normal hematopoiesis, OB-R was expressed in CD34(+) cells. Normal promyelocytes (CD34(-)33(+) and CD34(-)13(+)) expressed only very low levels of the short, presumably nonsignaling isoform. Both the long and short isoforms of OB-R were expressed in 10 of 22 samples from patients with newly diagnosed primary or secondary acute myeloid leukemia (AML), with a higher incidence of the long isoform in primary AML (87.6% v 28.6%; P =.01). The incidence of OB-R expression was higher in recurrent than in newly diagnosed AML (P <.001), and samples from four patients with refractory AML showed strong expression of both isoforms. Both OB-R isoforms were also expressed in newly diagnosed and recurrent acute promyelocytic leukemia cells but were essentially absent in samples of chronic or acute lymphocytic leukemia. In vitro growth of myeloid leukemic cell lines and of blasts from 14 primary AMLs demonstrated that recombinant human leptin alone induced low level proliferation, significantly (P <.05) increased proliferation induced by recombinant human granulocyte colony-stimulating factor, interleukin 3, and stem cell factor in a subset of AML and increased colony formation (P <.005). Also, leptin reduced apoptosis induced by cytokine withdrawal in MO7E and TF-1 cells. Serum leptin levels correlated only with body mass index (P <. 001) and gender (P =.03). Results confirm the reported expression of leptin receptor in normal CD34(+) cells and demonstrate the frequent expression of leptin receptors in AML blasts. While normal promyelocytes lack receptor expression, leukemic promyelocytes express both isoforms. We also demonstrate proliferative effects of leptin alone and in combination with other physiologic cytokines, and anti-apoptotic properties of leptin. These findings could have implications for the pathophysiology of AML. (+info)
Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.
(12/15103)
Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles. (+info)
Homotypic and heterotypic interaction of the neurofibromatosis 2 tumor suppressor protein merlin and the ERM protein ezrin.
(13/15103)
Ezrin, radixin and moesin (ERM) are homologous proteins, which are linkers between plasma membrane components and the actin-containing cytoskeleton. The ERM protein family members associate with each other in a homotypic and heterotypic manner. The neurofibromatosis 2 (NF2) tumor suppressor protein merlin (schwannomin) is structurally related to ERM members. Merlin is involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas, but the tumor suppressor mechanism is poorly understood. We have studied the ability of merlin to self-associate and bind ezrin. Ezrin was coimmunoprecipitated with merlin from lysates of human U251 glioma cells and from COS-1 cells transfected with cDNA encoding for merlin isoform I. The interaction was further studied and the association domains were mapped with the yeast two-hybrid system and with blot overlay and affinity precipitation experiments. The heterotypic binding of merlin and ezrin and the homotypic association of merlin involves interaction between the amino- and carboxy-termini. The amino-terminal association domain of merlin involves residues 1-339 and has similar features with the amino-terminal association domain of ezrin. The carboxy-terminal association domain cannot be mapped as precisely as in ezrin, but it requires residues 585-595 and a more amino-terminal segment. Unlike ezrin, merlin does not require activation for self-association but native merlin molecules can interact with each other. Heterodimerization between merlin and ezrin, however, occurs only following conformational alterations in both proteins. These results biochemically connect merlin to the cortical cytoskeleton and indicate differential regulation of merlin from ERM proteins. (+info)
Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis.
(14/15103)
Residues in annexin XI-A essential for binding of calcyclin (S100A6) were examined by site-directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI-A, GST-AXI 34-62 and GST-AXI 49-77 bound to calcyclin-Sepharose Ca2+-dependently. The mutants GST-AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST-AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the alpha-helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation. (+info)
Calcitonin gene-related peptide decreases expression of acetylcholinesterase in mammalian myotubes.
(15/15103)
Nerve-derived trophic factors are known to modulate expression of acetylcholinesterase (AChE) in skeletal muscle fibers, yet the precise identity of these factors remains elusive. In the present study, we treated mouse C2 myotubes with calcitonin gene-related peptide (CGRP). Compared to non-treated myotubes, cell-associated AChE activity levels were decreased by approximately 60% after 48 h of treatment. A parallel reduction in AChE total protein levels was also observed as determined by Western blot analysis. The reduction in AChE activity was due to a decrease in the levels of the G1 molecular form and to an elimination of G1. By contrast, levels of secreted AChE remained unchanged following CGRP treatment. Finally, the overall decrease in AChE activity was accompanied by a reduction in AChE transcripts which could not be attributed to changes in the transcriptional rate of the ACHE gene. (+info)
Complete exon-intron organization of the mouse fibulin-1 gene and its comparison with the human fibulin-1 gene.
(16/15103)
Fibulin-1 is a 90 kDa calcium-binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C-termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin-1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3' end. By searching the database we identified most of the exons encoding the human fibulin-1 gene and showed that its exon-intron organization is similar to that of the mouse gene. (+info)