Expression of cell adhesion molecule L1 in the long head of biceps tendon.

Several studies have proposed that the nervous system participates in nociception and tendon healing process. The neural cell adhesion molecule L1 (L1—CAM), which has an important role in neural development and nociceptive pathways, has been described in the past in the skeletal muscles and tendino—muscular junction. The role of this protein in tendon pathology is unknown. Here, we show that L1—CAM is expressed in human tendons. Samples of the long head of the biceps tendon (LHB) from six patients undergoing shoulder surgery were studied. Both Western blot and immunofluorescence revealed a strong expression pattern of L1—CAM. These L1—CAM positive cells were also Tuj1 positive, suggesting a neuronal origin. To our knowledge this is the first unequivocal evidence of the presence of L1 CAM in human tendons suggesting that it may play a role in organization of extracellular matrix and tendon pain.

Multiple influences on the migration of precerebellar neurons in the caudal medulla.

Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Hair cells and supporting cells share a common progenitor in the avian inner ear.

Sensory organs of the vertebrate inner ear contain two major cell types: hair cells (HCs) and supporting cells (SCs). To study the lineage relationships between these two populations, replication-defective retroviral vectors encoding marker genes were delivered to the otic vesicle of the chicken embryo. The resulting labeled clones were analyzed in the hearing organ of the chicken, called the basilar papilla (BP), after cellular differentiation. BPs were allowed to develop for 2 weeks after delivery of the retrovirus, were removed, and were processed histochemically as whole mounts to identify clones of cells. Clusters of labeled cells were evident in the sensory epithelium, the nonsensory epithelium, and in adjacent tissues. Labeled cell types included HCs, two morphologically distinct types of SCs, homogene cells, border cells, hyaline cells, ganglion cells, and connective tissue cells. Each clone was sectioned and cell-type identification was performed on sensory clones expressing retrovirally transduced beta-galactosidase. Cell composition was determined for 41 sensory clones, most of which contained both HCs and SCs. Clones containing one HC and one SC were observed, suggesting that a common progenitor exists that can remain bipotential up to its final mitotic division. The possibility that these two cell types may also arise from a mitotic precursor during HC regeneration in the mature basilar papilla is consistent with their developmental history.

The characterization, molecular cloning, and expression of a novel hematopoietic cell antigen from CD34+ human bone marrow cells.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

Isolation of the human MOX2 homeobox gene and localization to chromosome 7p22.1-p21.3.

We have isolated and characterized cDNA clones encoding a novel human homeobox gene, MOX2, the homologue of the murine mox-2 gene. The MOX2 protein contains all of the characteristic features of Mox-2 proteins of other vertebrate species, namely the homeobox, the polyhistidine stretch, and a number of potential serine/threonine phosphorylation sites. The homeodomain of MOX2 protein is identical to all other vertebrate species reported so far (rodents and amphibians). Outside the homeodomain, Mox-2 proteins share a high degree of identity, except for a few amino acid differences encountered between the human and the rodent polypeptides. A polyhistidine stretch of 12 amino acids in the N terminal region of the protein is also conserved among humans, rodents, and (only partly) amphibians. The chromosomal position of MOX2 was assigned to 7p22.1-p21.3.

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Localization of molluscan R15 alpha 2 peptide immunoreactivity in the mammalian brain.

The R15 neuropeptides have been identified in the marine mollusc Aplysia californica. They compose a new family of neuropeptides acting on the cardiovascular, digestive, respiratory, reproductive and nervous systems. In this report we show that one of the members of the R15 neuropeptide family, the alpha 2 peptide is conserved in lower mammals. We have identified R15 alpha 2 immunoreactive neurons in the neurosecretory cell groups of the hypothalamus and in the brainstem of the hedgehog (Erinaceus europaeus). The majority of labeled cells were localized to the anterior periventricular part of the paraventricular nucleus and the accessory neurosecretory cell groups in the lateral hypothalamus as well as to the dorsal part of the nucleus tractus solitarii. In the paraventricular nucleus, R15 alpha 2 immunoreactive neurons also exhibit immunoreactivity for oxytocin, corticotropin releasing factor, vasoactive intestinal polypeptide and for the FMRFamide-related peptide which we found to be conserved in the hedgehog brain as well. No complete colocalization of R15 alpha 2 with any of the neuroactive substances tested, is observed. The highest degree of coexistence occurs with FMRFamide-related peptide, followed by vasoactive intestinal polypeptide, oxytocin and corticotropin releasing factor.

Early arrest of B cell development in nude, X-linked immune-deficient mice.

Mice simultaneously expressing the nude and xid mutations have a severe deficit of both mature T and B cells. We now report studies designed to determine at which point in B cell differentiation development is arrested. Nude-xid mice have normal numbers of hematopoietic colony forming units (CFU-s) but lack two early pre-B cell markers: susceptibility to transformation by Abelson murine leukemia virus (A-MuLV) and production of cytoplasmic mu (C mu) heavy chain. Thus, there is a defect in lymphocyte development prior to or early in pre-B cell differentiation but after hematopoietic stem cell formation. The monoclonal reagents DNL 1.9, 14.8 and RA3-3A1/6.1, which react with the surface protein B220 (Ly5) on pro-B, C mu- pre-B, C mu+ pre-B, and surface Ig+ B cells, revealed the presence of positive cells in nude-xid mice. The bone marrow of nude-xid mice contains more B220+ cells than C mu+ cells. Our data suggest that the developmental block in these mice occurs at the earliest identifiable step in the B lymphocyte lineage, after the appearance of B220+ C mu- pro-B cells, but before the differentiation of C mu-bearing (pre-B) cells.