Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.     

Molecular cloning and DNA sequence analysis of genes encoding cytotoxic T lymphocyte-defined HLA-A3 subtypes: the E1 subtype

Influenza-specific cytotoxic T cells restricted by HLA-A3 and allogeneic CTL specific for HLA-A3 recognize differences between serologically indistinguishable HLA-A3 antigens. Previous biochemical studies have indicated that such differential recognition can be explained by alterations in the primary structure of class I heavy chains. Characterization of these sequence differences may therefore identify portions of the class I molecule that form determinants recognized by CTL. In this study, we describe the cloning and sequencing of an HLA-A3 subtype from donor E1 (E1-A3). Cloning of the gene encoding E1-A3 was simplified by determining that a 15.5-kb BamHI fragment contains the complete gene and is characteristic of HLA-A3 and only one other class I gene (HLA-A11). Comparison of the E1-A3 sequence to that of a previously sequenced HLA-A3 gene for exons encoding extracellular class I domains revealed three nucleotide differences. All of these differences were located within a discrete region of exon 3 (encoding the alpha 2 domain) and result in a change of two amino acids, at positions 152 (Glu----Val) and 156 (Leu----Gln). This finding suggests that these amino acids are crucial for the information of a determinant recognized by CTL. Furthermore, the altered nucleotide sequence of E1-A3 is identical to the sequence of the HLA-Aw24 gene for codons 128 to 161. These observations of multiple clustered changes in the E1-A3 subtype (relative to the prototype sequence) and identity of the altered sequence with the sequence of another class I gene support the concept that gene conversion is a primary mechanism for the generation of class I polymorphism.     

1,25-Dihydroxyvitamin D3 suppresses human T helper/inducer lymphocyte activity in vitro

The active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), suppresses in vitro immunoglobulin (Ig) production by activated peripheral blood mononuclear cells (PBM) from normal human subjects by inhibiting T helper/inducer TH cell activity. Normal PBM were fractionated into B, TH and T suppressor/cytotoxic (Ts) cells by fluorescence-activated cell sorting techniques. The resultant subsets were activated with mitogens and were cultured in the presence or absence of a receptor-saturating concentration of 1,25-(OH)2-D3. The sterol reduced [3H]thymidine incorporation in TH cells by 56%, with no effect on Ts or B cells. When 1,25-(OH)2-D3-treated TH cells were co-cultured with untreated B cells and culture supernatants assayed for Ig production, 1,25-(OH)2-D3 abrogated the inducing effect of TH cells on Ig synthesis by B cells. There was no inhibitory effect of the sterol on Ts or B cell activity. In addition, 1,25-(OH)2-D3 produced a dramatic inhibition of interleukin 2 (IL 2) production by activated PBM, but did not inhibit IL 2 receptor generation by these cells. Other vitamin D metabolites tested did not produce this effect. These results suggest that the TH lymphocyte is the specific cellular target for the immunoinhibitory effects of 1,25-(OH)2-D3.     

Hepatotoxic doses of dioxin do not damage mouse bone marrow chromosomes

We report on a study of the cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice of the C57B1/6J (with high-affinity TCDD receptor) or DBA/2J (with low-affinity TCDD receptor) strains were given single intraperitoneal injections of 50, 100 or 150 micrograms of TCDD/kg body weight. At various times (8-48 h) after injection, we examined bone marrow cells for cytogenetic effects by performing structural aberration, sister-chromatid exchange, and micronucleus tests. 1 month after exposure, liver sections were studied for hepatotoxic effects. We found no evidence of chromosome damage by TCDD given in doses that cause liver damage in both strains of mice.     

Reduced Apparent Photorespiration by the C(3)-C(4) Intermediate Species, Moricandia arvensis and Panicum milioides

The CO₂/O₂ specificity factor of sucrose gradient purified ribulose 1,5-bisphosphate carboxylase/oxygenase from the C₃-C₄ intermediate plants Moricandia arvensis (79 ± 1) and Panicum milioides (89 ± 2) was similar to the respective values of the enzyme from the closely related C₃ species, Moricandia foetida (80 ± 5) and Panicum laxum (86 ± 2). Thus, the kinetic properties of this bifunctional enzyme do not explain the reduced rates of photorespiration exhibited by either of these intermediate species.     

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.