Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53

Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.     

Molecular cloning of lymphadenopathy-associated virus

Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1-9.2 kilobases).     

The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus

Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.     

Viral transduction of c-myc gene in naturally occurring feline leukaemias

Feline leukaemia virus (FeLV) is epidemiologically associated with induction of the majority of lymphoid tumours of the domestic cat. However, about one-third of these tumours are devoid of exogenous virus or show evidence of virus integration only after tumour outgrowth. To help define the genetic mechanisms of feline lymphomagenesis we have explored here the possibility that cellular oncogenes (c-onc genes) are rearranged in tumour cell DNA. Of 16 FeLV-positive T-cell tumours among 31 naturally occurring lymphomas, 2 showed evidence of recombinant FeLV proviruses containing myc oncogene sequences. One of the two produced a transmissible myc-containing FeLV. In both cases c-myc and its surrounding DNA appeared unaltered. We believe that the association of myc with FeLV may result in its activation and play a part in the development of a significant fraction of cat T-cell lymphomas. Our findings contrast with studies of experimental induction of chicken lymphoma, in which myc activation occurs by retrovirus promoter insertion near c-myc (refs 3-5), rather than by incorporation into virus.     

Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.     

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.     

DNA sequence and expression of the B95-8 Epstein-Barr virus genome

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.     

Isolation of cDNA clones encoding T cell-specific membrane-associated proteins

Of 10 distinct cloned DNA copies of mRNAs expressed in T lymphocytes but not in B lymphocytes and associated with membrane-bound polysomes, one hybridizes to a region of the genome that has rearranged in a T-cell lymphoma and several T-cell hybridomas. These characteristics suggest that it encodes one chain of the elusive antigen receptor on the surface of T lymphocytes.