Abstract: Brain and immune system tissues express myelin basic protein (MBP) mRNAs that contain novel exons upstream of those of the classic MBPs. We have generated antisera against a recombinant protein that includes the deduced sequence for one of the predicted species of the new MBP‐related protein, hemopoietic HMBPR1. We report here the presence of multiple proteins reactive to the antisera in a range of tissues. The principal finding is that the antisera recognize a family of proteins of ∼25 kDa that are restricted to brain, thymus, and spleen, the only tissues in which the new MBP‐related transcripts are present. These antisera also detect other proteins of apparent molecular mass consistent with other isoforms of predicted MBP‐related proteins. The expression of MBP‐related proteins in immune system tissues may be important for self‐tolerance to CNS MBPs and the initiation of immune‐associated demyelinating diseases.
Abstract: The lck gene product, p56 lck , is a member of the src‐related family of protein tyrosine kinases. It is known as lymphocyte specific and involved in thymocyte development and in the immune response mediated by the T cell receptor. We report that the lck gene is also expressed in adult mouse CNS and that brain p56 lck is similar to the thymus protein. In situ hybridization and immunohistochemistry show that the lck gene is expressed in neurons throughout the brain in distinct regions, including hippocampus and cerebellum. In primary cultures from fetal mouse brain, neuronal cells are immunoreactive to Lck antiserum. This suggests that the lck gene product might be involved in a new signal transduction pathway in mouse brain.
ABSTRACT In quail oviduct, a 175K (K=103Mr) protein associated with striated rootlets was previously identified by Klotz and co-workers using monoclonal antibody CC310. As this monoclonal antibody recognizes several proteins on immunoblots of ciliated cells, we prepared a polyclonal antibody monospecific to the 175K protein by intrasplenic immunization of mice. Immunofluorescence study confirmed the distribution of the 175K protein at the apical part of the ciliated cell and its absence in other epithelial cells. Immunogold staining showed that this protein was strongly associated with the fibrillar axis of striated rootlets. The absence of labeling on striation suggested that rootlets were composed of several proteins, with one group forming the fibrillar axis and the second forming the striation. The formation of striated rootlets during ciliogenesis was studied using this polyclonal antibody. The 175K protein appeared at the beginning of centriologenesis in fibrillar material located around dense granules, and then around the generative complex. The formation of rootlets began at the basal pole of migrating basal bodies. The elongation of the rootlet axes took place when basal bodies were anchored to the plasma membrane.
Abstract A procedure adapted from that described by Mitchison and Kirschner [Nature 312:232–237, 1984] was used to isolate centrosomes from human lymphoid cells. High yields of homogeneous centrosomes (60% of the theoretical total, assuming one centrosome per cell) were obtained. Centrosomes were isolated as pairs of centrioles, plus their associated pericentriolar material. Ultrastructural investigation revealed: 1) a link between both centrioles in a centrosome formed by the gathering in of a unique bundle of thin filaments surrounding each centriole; 2) a stereotypic organization of the pericentriolar material, including a rim of constant width at the proximal end of each centriole and a disc of nine satellite arms organized according to a ninefold symmetry at the distal end and; 3) an axial hub in the lumen of each centriole at the distal end surrounded by some ill‐defined material. The total protein content was 2 to 3 × 10 −2 pg per isolated centrosome, a figure that suggests that the preparations were close to homogeneity. The protein composition was complex but specific, showing proteins ranging from 180 to 300 kD, one prominent band at 130 kD, and a group of proteins between 50 and 65 kD. Actin was also present in centrosome preparations. Functional studies demonstrated that the isolated centrosomes were competent to nucleate microtubules in vitro from purified tubulin in conditions in which spontaneous assembly could not occur. They were also very effective at inducing cleavage when microinjected into unfertilized Xenopus eggs.
Summary— The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre‐ and post‐embedding EM immunocytochemistry with a variety of anti‐tubulin antibodies. The 6‐11B‐1 monoclonal antibody, specific for the post‐translational acetylation of Lys 40 of α‐tubulin [40], and a polyclonal antibody raised against Paramecium axonemal tubulin (anti‐PA tubulin antibody) [16] both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti‐PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti‐tubulin antibody and the anti‐PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti‐PA tubulin one. This provided a strong indication that the anti‐PA tubulin antibody is directed against a post‐translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol‐treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti‐PA tubulin antibody however, suggesting that in Metazoa the post‐translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post‐translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.
CD4 is a member of the Ig gene super family expressed on the surface of many thymocytes and of a subset of T lymphocytes. Human CD4 is the receptor for HIV envelope glycoprotein gp120. Human and mouse CD4 transcripts are expressed in human and mouse central nervous system (CNS), but no corresponding proteins have been reported yet. We have analyzed mRNA expression and carried out immunological experiments on adult mouse brain with probes specific for the long and short CD4 transcripts and with antibodies monospecific for mouse CD4. The main result of these experiments is that the full length CD4 transcript and the CD4 protein are expressed coordinately in neurons throughout the adult mouse brain. CD4 immunoreactivity is also present in brain small vessel walls, ependymal cells, and choroid plexus. The brain mouse CD4 protein is indistinguishable from the thymus protein. In addition, we show that neuronal cells in primary cultures from human fetal CNS are immunoreactive to human CD4 mAbs.
A spontaneously arising rabbit anti-centrosome serum with strong human specificity, used to identify specific antigens in isolated centrosomes, was shown to react with several noncentrosomal proteins including a 36-kDa protein that appeared to be the major cellular antigen. To explore the immunological relationship between noncentrosomal and centrosomal antigens, immunoglobulins were affinity purified using the individual noncentrosomal antigens (from lymphoblastoma KE37 cells) and were tested for their capacity to bind to human centrosomes in situ and to proteins from isolated centrosomes. In this way, the 36-kDa antigen, an abundant cytosolic protein, was shown to share at least one antigenic determinant with high molecular weight centrosomal proteins. This antigen was further identified by mild proteolysis as the glycolytic enzyme lactate dehydrogenase. In all the analyzed human cell lines, the centrosomal staining in situ was correlated with a strong labeling of purified lactate dehydrogenase in immunoblots. Conversely, the absence of centrosomal staining in rodent cells was always correlated with the absence of lactate dehydrogenase labeling. These data suggest an evolutionary relationship between centrosomal proteins and this "housekeeping" enzyme.
