A molecular defect in thrombasthenic platelets.

An IgG antibody found in the serum of a thrombasthenic patient reacted in complement fixation with platelets from 350 normal individuals but was nonreactive with platelets from eight other thrombasthenic patients. ADP-induced aggregation of normal platelets was inhibited by the patient's antibody. Family studies using the quantitative complement fixation test showed that healthy heterozygotes were easily distinguishable from normal or thrombasthenic individuals since their platelets had an intermediate amount of the reactive antigen. Indirect immunoprecipitation tests using this serum and soluble membrane antigens labeled with iodine-125 that had been extracted from normal platelets by the detergent Nonidet P-40 gave a single radioactive peak at 120,000 mol wt in sodium dodecyl sulfate polyacrylamide gel electrophoresis. A similar estimate of the molecular weight was obtained from Sephadex G-200 filtration of the soluble antigens extracted from normal platelets by spontaneous release or chaotropic agents and tested in complement fixation with the patient's serum. These findings strongly suggest that the molecule recognized by this antibody is absent or structurally modified in thrombasthenia cases and that it may be involved in platelet aggregation.

Molecular data on urinary glycoproteins with human leucocyte antigen (HLA) activity.

Two glycoproteins characterized by their serological activities (HLA-A9 and HLA-B12), their isoelectric points and their molecular weights were purified from urine from a patient suffering from tubular proteinuria (cystinosis). Their physicochemical properties as well as an important increase of their specific activities during the different purification steps suggested that they behave as human leucocyte antigens (HLA) which had been excreted into urine. Their amino acid compositions and N-terminal sequences were different to those described for HLA solubilized from cultured human lymphoblast cell lines. The N-terminal sequences of the two serologically active glycoproteins were identical to the N-terminal sequence of another recently purified human urinary glycoprotein called human complex-forming glycoprotein. The relationship between HLA, human complex-forming glycoprotein and the serologically active urinary glycoproteins is discussed.

alpha 1-Microglobulin from normal and pathological urines.

alpha 1-Microglobulin was purified from normal and pathological urines. Significant differences were found in the amino acid compositions of the alpha 1-microglobulin isolated from these two sources. In addition electrofocusing of alpha 1-microglobulin from normal urine gave rise to two peaks of equal intensity with rather acidic isoelectric points (3.8 and 4.2), whilst alpha 1-microglobulin from pathological urine showed two peaks in a 1:5 ratio with less acidic isoelectric points (4.2 and 4.7). Further charge heterogeneity was also observed in the second peaks from both sources. The sugar compositions were also established, as well as the N-terminal sequences of the alpha 1-microglobulin of both peaks isolated from normal and pathological urines.

Molecular cloning and nucleotide sequence of a cDNA clone coding for rat brain myelin proteolipid.

A cDNA library from rat brain was constructed in pBR322 and screened with a 14-mer mixed oligonucleotide probe based on residues 231-235 of bovine proteolipid (PLP). A positive clone was isolated: it contained a 1334-base-pair cDNA insert and was subjected to DNA sequence analysis. The cDNA encoded information for the 276 amino acids of rat PLP. Comparison with bovine PLP sequence showed a complete amino acid sequence homology except for 4 amino acid residues.

Human platelets as a source of HL-A antigens : a study of various solubilization techniques.

The efficiency of various methods of solubilizing HL-A platelet antigens was investigated. The yield of soluble material was compared with that obtained from lymphocytes in culture in order to judge the quality of platelets as a source of HL-A antigens. The conclusion was reached that platelets, easily obtainable, can be considered as a good source of HL-A antigens.

Developmental expression of myelin proteolipid, basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase transcripts in different rat brain regions.

RNA was extracted from five different rat brain regions during development, starting from embryonic day 15 (E15) until postnatal day 60 (P60). These RNA preparations were analyzed by both Northern and dot blot for their content of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), myelin proteolipid protein (PLP), and myelin basic protein (MBP) -specific transcripts. CNPase mRNA was readily detectable at E15 and PLP mRNA at P1 in all brain regions examined. In contrast, expression of MBP mRNA followed a caudorostral gradient. It was first observed at P1 in the mesencephalon and at P9-P11 in the olfactory bulb. Expression of these three transcripts displayed two types of developmental profiles. One was termed biphasic because the specific mRNA level increased regularly and then reached a plateau level. The other developmental profile was termed triphasic, because there was a gradual increase in the level of specific transcripts with a sudden appearance of a sharp peak followed by a decline to a plateau level. When the triphasic pattern was observed, the date of the peak appearance was probe-, but not region-, dependent. It was P15 for CNPase, P18 for MBP, and P21 for PLP. As these peaks occurred at a time during development when myelination was the most active, we postulate the existence of a transient external signal, perhaps neuronal, which would be responsible for this increased amount of myelin-related transcripts.

Proteolipid DM-20 predominates over PLP in peripheral nervous system.

The major central nervous system (CNS) myelin proteolipid (PLP) is also expressed in the peripheral nervous system (PNS). This paper gives evidence that DM-20, an isoform of PLP, also occurs in rat sciatic nerves, where, in contrast to what is seen in CNS myelin, it predominates over PLP. This conclusion was reached on the basis of results obtained by immunoblot analysis of a crude proteolipid extract from adult peripheral nerve with two site-specific anti-proteolipid (PLP and DM-20) antibodies. This finding was further corroborated by characterization of the products obtained by Polymerase Chain Reaction (PCR) amplification of cDNAs synthesized from total RNA of 14-day-old sciatic nerves. The significance of the occurrence of these proteolipids in PNS remains obscure.

Characterization of alpha 1-microglobulin in human colostrum and milk.

The mean concentration of alpha 1-microglobulin in human colostrum and milk, estimated by electroimmunoassay, was found to be about 0.4-0.6 mg/l and 0.1-0.2 mg/l, respectively. Both liquids contained alpha 1-microglobulin in mono- and dimeric forms, while the presence of higher polymeric forms, as characterized in plasma, could not be demonstrated.

Myelin proteolipid protein (PLP and DM-20) transcripts are deleted in jimpy mutant mice.

The myelin-associated proteolipid protein, PLP, is one of the two major components of the central nervous system (CNS) myelin. We analyze, by using a rat PLP cDNA and S1 nuclease protection experiments, the PLP transcripts in the mouse brain and show that the PLP gene encodes two different but related mRNA transcripts, the PLP and the DM-20 transcripts. On the other hand, we demonstrate that in the jimpy mutant, which is characterized by an abnormal CNS myelination, both these transcripts are partially deleted in the 3' end of their coding region. The deletion is 70 (+/- 5) nucleotides long. Implications of this finding for the synthesis of PLP and DM-20 proteins in the mutant are discussed.

Major Myelin proteolipid: the 4-alpha-helix topology.

Several conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP). We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology. A new, simple model is suggested, which features four hydrophobic alpha-helices spanning the whole thickness of the lipid bilayer. Its orientation may be such that both the N- and C-termini face the cytosol. None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model. The effect or absence thereof of various PLP mutations is discussed in the frame of the proposed 4-helix topology.

Spontaneous release of soluble HL-A antigens from platelets during conservation.

Experiments with the aim of studying the solubilisation of HL-A antigens from blood platelets by methods which do not involve any biologically active processes (moderate, discontinuous agitation of a low concentration of platelets suspended in a saline medium, in the presence of an antiseptic; supernatants collected at frequent intervals) have shown that platelets release membrane proteins, including HL-A antigens, spontaneously. Optimal conditions for the treatment of membrane proteins have been perfected. The great stability of HL-A antigens under these conditions permits prolonged treatment. The products extracted are soluble and extremely complex. The molecular weight of the HL-A antigens is between 40,000 and 70,000.

Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene.

We have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5' flanking region reveals that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerase chain reaction method.

Functional analysis of the mouse myelin/oligodendrocyte glycoprotein gene promoter in the oligodendroglial CG4 cell line.

Myelin/oligodendrocyte glycoprotein (MOG) is a late phylogenetic acquisition among vertebrates that is found only in mammals. MOG is a minor component of myelin protein, representing approximately 0.01-0.05% of the total. Regulatory elements in the MOG gene were identified by transfecting the oligodendroglial CG4 cell line with chimeric MOG-luciferase genes. Only a few hundred base pairs upstream of the coding sequence were necessary for high-level activity of the mouse MOG promoter. More distal recognition sites may exist, because silencing activity, indicative of negative regulatory elements, was detected upstream of base pair 657. Transcriptional activity of chimeric MOG- and myelin basic protein-luciferase genes was greater in CG4 cells than in 3T3 fibroblasts or C6 glioblastoma, demonstrating their superiority for functional analysis of myelin gene regulatory elements.

Alexander disease: putative mechanisms of an astrocytic encephalopathy.

Alexander disease (AXD) is the first primary astrocytic disorder. This encephalopathy is caused by dominant mutations in the glial fibrillary acidic protein (GFAP) gene, encoding the main intermediate filament of astrocyte. Pathologically, this neurodegenerative disease is characterised by dystrophic astrocytes containing intermediate filament aggregates associated with myelin abnormalities. More than 20 GFAP mutations have been reported. Many of them cluster in highly conserved regions between several intermediate filaments. Contrary to other intermediate filament-related diseases, AXD seems to be the consequence of a toxic gain of function induced by aggregates. This is supported by the phenotype of mice overexpressing human GFAP. Nevertheless, GFAP null mice display myelin abnormalities and blood-brain barrier dysfunction that are present in AXD. Given the pivotal role of astrocytes in brain physiology, there are many possibilities for astrocytes to dysfunction and to impair the functions of other cells. Physiopathological hypotheses are discussed in the frame of AXD.

Cell-free synthesis of hemocyanin from the scorpion Androctonus australis. Characterization of the translation products by monospecific antisera.

Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8-10) of 70-73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti-(dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace.