[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Linkage relationships of seven enzyme and two pigmentation loci in the snail Biomphalaria glabrata

Crossing experiments with inbred stocks of the snail (Biomphalaria glabrata) demonstrated that variants at two loci determining pigmentation and seven enzyme-determining loci exhibited normal Mendelian segregation ratios in F2 progeny. Among 39 pairwise comparisons for joint segregation, there was evidence of genetic linkage between a locus controlling mantle pigmentation (S) and 6-phosphogluconate dehydrogenase (Pgd) and confirmation of a previously described linkage between esterase-2 (Est-2) and catalase (Cat). Recombination fractions were estimated to be 17 +/- 4 for S-Pgd and 33 +/- 5 for Est-2-Cat. The remaining five loci--Acon-1, Pgm-1, Lap-1, Lap-2, and Pgd--assorted independently. This brings to 17 the number of loci examined for segregation and assortment in this medically important species. As Biomphalaria has a chromosome number n = 18, markers should soon be available for most or all of the linkage groups.     

The cytolytic and regulatory role of natural killer cells in experimental neoplasia

NK cells are defined here as cells, other than macrophages and polymorphonuclear leucocytes, from non-immunized animals (or humans) which are cytotoxic for neoplastic and non-neoplastic targets in the absence of specific antibody. Though not requiring antibody, they may function as K cells in ADCC. This definition includes cells activated nonspecifically by such agents as IFN and IL-2. Murine NK cells may be subdivided into two types by differences in the kinetics of target-cell lysis. Those we label Type 1 correspond roughly to what others have called NKA, NKL or simply NK cells; those of Type 2 to NKB, NKS and NC cells. Type 1 cells express various antigens, including NK-1, Thy-1 (50%), Ly-1 (25%), Qa-3, Qa-4, Qa-5, Ly-5, Ly-6, Ly-10, Ly-11 and asialo-GM1, not expressed by Type 2 cells, whereas Mac-1 may be expressed by both types. At least some NK cells appear to be pre-thymic cells which, in the presence of a thymus, can differentiate into T cells. The level of NK activity is influenced by the age and genetic background of the mouse, the organ from which the cells are obtained, and a variety of experimental manipulations. Type 1 activity is increased by IFN and IL-2; Type 2 activity by IL-3. IFN appears to be concerned in the development of spontaneous NK activity in young mice. Many experiments have shown that NK cells may inhibit the growth of tumours which are sensitive to NK cells of the same type in vitro. Inhibitory cells which suppress NK activity may play an important regulatory role in vivo. There is still uncertainty about how NK cells recognize their targets. Possibilities discussed are: (1) specific interacting molecules; (2) more diffuse properties of target cell membranes; (3) absence of MHC-coded self-recognition markers. Certainly, the presence of a Class 1 MHC molecule is not necessary. NK killing appears to be mediated by cytotoxins released by NK cells. In vivo, NK cells contribute to limiting the development of transplanted and primary tumours, and metastasis from established tumours. NK cells seem well qualified to act as a first-line defence against neoplasia, and may kill cells not killed by T cells. Transfer of NK cells may be of value in the treatment of cancer.     

Population genetics of Biomphalaria straminea in Hong Kong. A neotropical schistosome-transmitting snail recently introduced into China

Since its introduction in the early 1970s Biomphalaria straminea (Gastropoda: Planorbidae) has spread rapidly and is locally the most abundant fresh water snail in Hong Kong. Studies of 19 electrophoretically detected loci in four populations show that the colonists retain high levels of variability (P = 0.26, H = 0.056 - 0.097), comparable with those found in autochthonous samples of related species. Genotype frequencies at the five polymorphic loci, and a comparison of maternal and progeny genotypes of individual field-collected snails, revealed no evidence for self-fertilization in these functional hermaphrodites. F statistics indicated minimal genetic structuring, presumably because of outcrossing and recency of origin of the populations. Geographic distribution of various alleles and their frequencies suggest that two southern populations were derived from the original colonists by dispersal but that a northern population represents a second introduction in about 1982. This interpretation (based on genetics) is consistent with the known history of the various populations. The Asian populations of this South American snail are interpreted as being in the "flush" phase of the colonization process. Finally, the probability of the secondary spread of this snail from Hong Kong, and the probability of its parasite, the human blood fluke Schistosoma mansoni, being introduced to Asia are discussed.     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.