Myosin subfragment-1 is sufficient to move actin filaments in vitro

The rotating crossbridge model for muscle contraction proposes that force is produced by a change in angle of the crossbridge between the overlapping thick and thin filaments. Myosin, the major component of the thick filament, is comprised of two heavy chains and two pairs of light chains. Together they form two globular heads, which give rise to the crossbridge in muscle, and a coiled-coil rod, which forms the shaft of the thick filament. The isolated head fragment, subfragment-1 (S1), contains the ATPase and actin-binding activities of myosin (Fig. 1). Although S1 seems to have the requisite enzymatic activity, direct evidence that S1 is sufficient to drive actin movement has been lacking. It has long been recognized that in vitro movement assays are an important approach for identifying the elements in muscle responsible for force generation. Hynes et al. showed that beads coated with heavy meromyosin (HMM), a soluble proteolytic fragment of myosin consisting of a part of the rod and the two heads, can move on Nitella actin filaments. Using the myosin-coated surface assay of Kron and Spudich, Harada et al. showed that single-headed myosin filaments bound to glass support movement of actin at nearly the same speed as intact myosin filaments. These studies show that the terminal portion of the rod and the two-headed nature of myosin are not required for movement. To restrict the region responsible for movement further, we have modified the myosin-coated surface assay by replacing the glass surface with a nitrocellulose film. Here we report that myosin filaments, soluble myosin, HMM or S1, when bound to a nitrocellulose film, support actin sliding movement (Fig. 2). That S1 is sufficient to cause sliding movement of actin filaments in vitro gives strong support to models of contraction that place the site of active movement in muscle within the myosin head.     

Localization of the gene for familial adenomatous polyposis on chromosome 5

Colorectal cancer is the second most common cancer in the United Kingdom and other developed countries in the West. Although it is usually not familial, there is a rare dominantly inherited susceptibility to colon cancer, familial adenomatous polyposis (FAP; also often previously called familial polyposis coli). During adolescence affected individuals develop from a few hundred to over a thousand adenomatous polyps in their large bowel. These are sufficiently likely to give rise to adenocarcinomas to make prophylactic removal of the colon usual in diagnosed FAP individuals. Adenomas may occur elsewhere in the gastrointestinal tract and the condition is often associated with other extracolonic lesions, such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours. Adenomata have been suggested to be precancerous states for most colorectal tumours. Knudson has suggested that the mutation for a dominantly inherited cancer susceptibility may be the first step in a recessive change in the tumour cells, and that the same gene may be involved in both familial and non-familial cases of a given tumour. Following up a case report of an interstitial deletion of chromosome 5 in a mentally retarded individual with multiple developmental abnormalities and FAP, we have now shown that the FAP gene is on chromosome 5, most probably near bands 5q21-q22.     

A new immunomodulating compound (AS-101) with potential therapeutic application

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).     

Millisecond X-ray diffraction and the first electron density map from Laue photographs of a protein crystal

Attempts to use X-ray crystallography to extract three-dimensional information on transient phenomena in crystals have been hampered primarily by long data collection times. Here we report on the first difference Fourier map obtained from Laue diffraction photographs of a protein crystal, glycogen phosphorylase b. Data collection time was 3 s using the high-intensity white X-radiation generated on the wiggler magnet of the Daresbury Synchrotron Radiation Source (SRS), but data acquisition in the millisecond-submillisecond range is possible. The method presented here uses a simple difference technique and was designed to analyse structural changes relative to a known starting structure. The combination of this approach with cine techniques allows the recording of three-dimensional motion pictures at atomic resolution and opens up new areas in structural biology and chemistry.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene

Genes involved in the transduction of signals required for normal cell proliferation commonly appear to be subverted in the neoplastic process. One such group is the highly conserved family of ras genes, which have been detected as transforming genes in a wide variety of naturally occurring tumours. By analogy with other known G proteins, the p21 proteins encoded by ras genes may act as regulatory proteins in the transduction of signals that lead to DNA synthesis. A major pathway involved in the DNA synthesis induced by growth factors is mediated by phosphatidylinositol turnover: cleavage of phosphoinositides by phospholipase C produces 1,2-diacylglycerol, and inositol phosphates. The former acts as an essential cofactor for protein kinase C (ref. 4), and inositol-(1,4,5)-triphosphate mobilizes Ca2+ from non-mitochondrial intracellular stores. We demonstrate a reproducible increase in 1,2-diacylglycerol, in the absence of a detectable increase in inositol phosphates, in transformed cells containing Ha-ras oncogenes and with different membrane targeting signals for the ras p21 protein. These findings suggest that a source other than phosphoinositides exists for the generation of 1,2-diacylglycerol and that the Ha-ras oncogene specifically activates this novel pathway for 1,2-diacylglycerol production.     

AIDS virus-specific cytotoxic T lymphocytes in lung disorders

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.     

The role of clonal selection and somatic mutation in autoimmunity

Polyclonal activation has been proposed as the reason that autoantibodies are produced during autoimmune disease. This model denies a role for specific antigen selection of B cells and predicts instead a multiclonal population of unmutated or randomly mutated autoantibodies. We have found that the genetic features and clonal composition of spontaneously derived immunoglobulin G (IgG) antiself-IgG (rheumatoid factor (RF] autoantibodies derived from the autoimmune MRL/lpr mouse strain are inconsistent with both the predictions of this model and the actual outcome of experimental polyclonal activation. Instead we have found that MRL/lpr RFs are oligoclonal or even monoclonal in origin. They harbour numerous somatic mutations which are distributed in a way that suggests immunoglobulin-receptor-dependent selection of these mutations. In this sense, the MRL/lpr RFs resemble antibodies elicited by exogenous antigens after secondary immunization. The parallels suggest that, like secondary immune responses, antigen stimulation is important in the generation of MRL/lpr RFs.